Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors

ABSTRACT

The present invention is directed to bispecific, heterodimeric immunomodulatory antibodies.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.16/184,929, filed Nov. 8, 2018 which claims priority to U.S. PatentApplication Nos. 62/583,431, filed Nov. 8, 2017; 62/614,271, filed Jan.5, 2018; 62/640,468, filed Mar. 8, 2018; 62/683,554, filed Jun. 11, 2018and 62/724,502; filed Aug. 29, 2018, all of which are expresslyincorporated herein by reference in their entirety, with particularreference to the figures, legends, and claims therein

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 16, 2021, isnamed 067461-5216-US_SL.txt and is 28,257,248 kilobytes in size.

BACKGROUND OF THE INVENTION

Tumor-reactive T cells lose their cytotoxic ability over time due toup-regulation of inhibitory immune checkpoints such as PD-1 and CTLA-4.Two parallel therapeutic strategies are being pursued for de-repressingtumor-reactive T cells so that they can continue to kill tumor cells.

The first approach is immune immunomodulatory blockade by treating withantagonistic monoclonal antibodies that bind to either theimmunomodulatory itself (PD-1, CTLA-4, etc.) or its ligand (PD-L1,PD-L2, CD80, CD86, etc.), thus removing the inhibitory signals holdingback tumor-reactive T cells from tumor cell killing. Immunomodulatoryreceptors such as CTLA-1, PD-1 (programmed cell death 1), TIM-3 (T cellimmunoglobulin and mucin domain 3), LAG-3 (lymphocyte-activation gene3), TIGIT (T cell immunoreceptor with Ig and ITIM domains), and others,inhibit the activation, proliferation, and/or effector activities of Tcells and other cell types. Guided by the hypothesis thatimmunomodulatory receptors suppress the endogenous T cell responseagainst tumor cells, preclinical and clinical studies of anti-CTLA4 andanti-PD1 antibodies, including nivolumab, pembrolizumab, ipilimumab, andtremelimumab, have indeed demonstrated that immunomodulatory blockaderesults in impressive anti-tumor responses, stimulating endogenous Tcells to attack tumor cells, leading to long-term cancer remissions in afraction of patients with a variety of malignancies. Unfortunately, onlya subset of patients responds these therapies, with response ratesgenerally ranging from 10 to 30% and sometimes higher for eachmonotherapy, depending on the indication and other factors.

The second approach for de-repressing tumor-reactive T cells is T cellcostimulation by treating with agonistic antibodies that bind tocostimulatory proteins such as ICOS, thus adding a positive signal toovercome the negative signaling of the immune checkpoints.

Accordingly, the invention is directed to bispecific antibodies thatbind to costimulatory receptors (e.g. ICOS, GITR, OX40, 4-1BB) as wellas checkpoint receptors (e.g. PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, BTLAand TIGIT.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides bispecific antibodiesthat monovalently binds a human costimulatory receptor and monovalentlybinds a human checkpoint receptor for use in activating T cells for thetreatment of cancer.

In some aspects, the costimulatory receptor is selected from the groupconsisting of ICOS, GITR, OX40 and 4-1BB.

In additional aspects, the checkpoint receptor is selected from thegroup consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIGIT and TIM-3.

In further aspects, the antibody binds an antigen pair selected from:ICOS and PD-1, ICOS and CTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS andPD-L1, ICOS and BTLA, ICOS and TIGIT, GITR and TIGIT, GITR and PD-1,GITR and CTLA-4, GITR and LAG-3, GITR and TIM-3, GITR and PD-L1, GITRand BTLA, OX40 and PD-1, OX40 and TIGIT, OX40 and CTLA-4, IC OX40 OS andLAG-3, OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB and PD-1,4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB and PD-L1,TIGIT and 4-1BB and 4-1BB and BTLA.

In additional aspects, the bispecific antibody has a format selectedfrom those outlined in FIG. 2.

In further aspects, the invention provides heterodimeric antibodiescomprising: a) a first heavy chain comprising a first Fc domain, anoptional domain linker and a first antigen binding domain comprising anscFv that binds a first antigen; b) a second heavy chain comprising aheavy chain comprising a heavy chain constant domain comprising a secondFc domain, a hinge domain, a CH1 domain and a variable heavy domain; andc) a light chain comprising a variable light domain and a light chainconstant domain; wherein said variable heavy domain and said variablelight domain form a second antigen binding domain that binds a secondantigen, wherein one of said first and second antigen binding domainsbinds human ICOS and the other binds human PD-1.

In an additional aspect, the invention provides heterodimeric bispecificantibodies comprising: a) a first heavy chain comprising: i) a firstvariant Fc domain; and ii) a single chain Fv region (scFv) that binds afirst antigen, wherein said scFv region comprises a first variable heavychain, a variable light chain and a charged scFv linker, wherein saidcharged scFv linker covalently attaches said first variable heavy chainand said variable light chain; and b) a second heavy chain comprising aVH—CH1-hinge-CH2-CH3 monomer, wherein VH is a second variable heavychain and CH2-CH3 is a second variant Fc domain; and c) a light chain;wherein said second variant Fc domain comprises amino acid substitutionsN208D/Q295E/N384D/Q418E/N241D, wherein said first and second variant Fcdomains each comprise amino acid substitutionsE233P/L234V/L235A/G236del/S267K; and wherein said first variant Fcdomain comprises amino acid substitutions S364K/E357Q and second variantFc domain comprises amino acid substitutions L368D/K370S, wherein one ofsaid first and second antigen binding domains binds human ICOS and theother binds human PD-1, and wherein numbering is according to the EUindex as in Kabat.

In some aspects the heterodimeric antibodies have first and secondvariant Fc domains that each comprise M428L/N434S.

In an additional aspect, the invention provides heterodimeric antibodiescomprising: a) a first heavy chain comprising: i) a first variant Fcdomain; and ii) a single chain Fv region (scFv) that binds a firstantigen, wherein said scFv region comprises a first variable heavychain, a variable light chain and a charged scFv linker, wherein saidcharged scFv linker covalently attaches said first variable heavy chainand said variable light chain; and b) a second heavy chain comprising aVH—CH1-hinge-CH2-CH3 monomer, wherein VH is a second variable heavychain and CH2-CH3 is a second variant Fc domain; and c) a light chain;wherein said first and second variant Fc domains comprises a set ofheterodimerization variants selected from the group consisting ofL368D/K370S: S364K/E357Q; L368D/K370S: S364K; L368E/K370S: S364K;T411E/K360E/Q362E: D401K; and T366S/L368A/Y407V: T366W, and wherein oneof said first and second antigen binding domains binds human ICOS andthe other binds human PD-1, and wherein numbering is according to the EUindex as in Kabat.

In a further aspect, the invention provides nucleic acid compositionscomprising nucleic acids that encode the heterodimeric antibodies of theinvention, expression vector compositions comprising the nucleic acids,and host cells comprising the expression vector compositions.

In an additional aspect, the invention provides heterodimeric antibodiesfor use in the activation of T cells for the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents expression data (RNAseq V2 RSEM) of PD-1 and T cellcostimulatory receptors for bladder, breast, colon, head & neck, kidney,lung-adeno, lung squamous, ovarian, pancreatic, prostate, and melanomacancer compiled from The Cancer Genome Atlas (TCGA). The square of thePearson correlation coefficient was calculated for PD-1 against T cellcostimulatory receptors.

FIG. 2A to 2N depict several formats for the bispecific antibodies ofthe present invention. The first is the “bottle opener” format, with afirst and a second anti-antigen binding domain. Additionally, mAb-Fv,mAb-scFv, Central-scFv, Central-Fv, one armed central-scFv, onescFv-mAb, scFv-mAb dual scFv format are all shown. FIG. 2J depicts the“central-scFv2” format, with two Fab-scFv arms, wherein the Fabs bind afirst antigen and the scFvs bind a second antigen. FIG. 2K depicts thebispecific mAb format, with a first Fab arm binding a first antigen anda second Fab arm binding a second antigen. FIG. 2L depicts the DVD-IgGformat (see, e.g., U.S. Pat. No. 7,612,181, hereby expresslyincorporated by reference and as discussed below). FIG. 2M depicts theTrident format (see, e.g., WO 2015/184203, hereby expressly incorporatedby reference and as discussed below). For all of the scFv domainsdepicted, they can be either N- to C-terminus variable heavy-(optionallinker)-variable light, or the opposite. In addition, for the one armedscFv-mAb, the scFv can be attached either to the N-terminus of a heavychain monomer or to the N-terminus of the light chain.

FIGS. 3A and 3B depicts the sequences of XENP23104, a bottle openerformat with the ICOS as the Fab side ([ICOS]_H0.66_L0) and the PD-1 asthe scFv (1G6_L1.94_H1.279), and includes the M428L/434S variant toextend serum half life. The CDRs are underlined, the scFv linker isdouble underlined (in the sequences, the scFv linker is a positivelycharged scFv (GKPGS)4 linker (SEQ ID NO: 26209), although as will beappreciated by those in the art, this linker can be replaced by otherlinkers, including uncharged or negatively charged linkers, some ofwhich are depicted in FIG. 8), and the slashes indicate the border(s) ofthe variable domains. In addition, the naming convention illustrates theorientation of the scFv from N- to C-terminus; some of the sequencesherein are oriented as VH-scFv linker-VL (from N- to C-terminus), whilesome are oriented as VL-scFv linker-VH (from N- to C-terminus), althoughas will be appreciated by those in the art, these sequences may also beused in the opposition orientation from their depiction herein. As notedherein and is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table 1, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems.

FIG. 4A to 4F depict useful pairs of heterodimerization variant sets(including skew and pI variants). On FIGS. 4C and F, there are variantsfor which there are no corresponding “monomer 2” variants; these are pIvariants which can be used alone on either monomer, or included on theFab side of a bottle opener, for example, and an appropriate chargedscFv linker can be used on the second monomer that utilizes a scFv asthe second antigen binding domain. Suitable charged linkers are shown inFIG. 8.

FIG. 5 depict a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants of theinvention (and other variant types as well, as outlined herein).

FIG. 6 depict useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants). Generally,ablation variants are found on both monomers, although in some casesthey may be on only one monomer.

FIGS. 7A and 7B show two particularly useful embodiments of theinvention. The “non-Fv” components of this embodiment is shown in FIG.9A, although the other formats of FIG. 9 can be used as well.

FIGS. 8A and 8B depict a number of charged scFv linkers that find use inincreasing or decreasing the pI of heterodimeric antibodies that utilizeone or more scFv as a component. The (+H) positive linker findsparticular use herein. A single prior art scFv linker with single chargeis references as “Whitlow”, from Whitlow et al., Protein Engineering6(8):989-995 (1993). It should be noted that this linker was used forreducing aggregation and enhancing proteolytic stability in scFvs.

FIGS. 9A to 9D shows the sequences of several useful bottle openerformat backbones based on human IgG1, without the Fv sequences (e.g. thescFv and the vh and vl for the Fab side). Bottle opener backbone 1 isbased on human IgG1 (356E/358M allotype), and includes the S364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421D pI variantson the Fab side and the E233P/L234V/L235A/G236del/S267K ablationvariants on both chains. Bottle opener backbone 2 is based on human IgG1(356E/358M allotype), and includes different skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains. Bottleopener backbone 3 is based on human IgG1 (356E/358M allotype), andincludes different skew variants, the N208D/Q295E/N384D/Q418E/N421D pIvariants on the Fab side and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Bottle opener backbone 4 is based onhuman IgG1 (356E/358M allotype), and includes different skew variants,the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains. Bottleopener backbone 5 is based on human IgG1 (356D/358L allotype), andincludes the S364K/E357Q: L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains. Bottleopener backbone 6 is based on human IgG1 (356E/358M allotype), andincludes the S364K/E357Q: L368D/K370S skew variants,N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as an N297A variant on both chains. Bottle opener backbone 7 isidentical to 6 except the mutation is N297S. Alternative formats forbottle opener backbones 6 and 7 can exclude the ablation variantsE233P/L234V/L235A/G236del/S267K in both chains. Backbone 8 is based onhuman IgG4, and includes the S364K/E357Q: L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains, aswell as a S228P (EU numbering, this is S241P in Kabat) variant on bothchains that ablates Fab arm exchange as is known in the art. Alternativeformats for bottle opener backbone 8 can exclude the ablation variantsE233P/L234V/L235A/G236del/S267K in both chains Backbone 9 is based onhuman IgG2, and includes the S364K/E357Q: L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side. Backbone 10is based on human IgG2, and includes the S364K/E357Q: L368D/K370S skewvariants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab sideas well as a S267K variant on both chains.

As will be appreciated by those in the art and outlined below, thesesequences can be used with any vh and vl pairs outlined herein, with onemonomer including a scFv (optionally including a charged scFv linker)and the other monomer including the Fab sequences (e.g. a vh attached tothe “Fab side heavy chain” and a vl attached to the “constant lightchain”). That is, any Fv sequences outlined herein for anti-CTLA-4,anti-PD-1, anti-LAG-3, anti-TIM-3, anti-TIGIT and anti-BTLA, whether asscFv (again, optionally with charged scFv linkers) or as Fabs, can beincorporated into these FIG. 37 backbones in any combination. Theconstant light chain depicted in FIG. 9A can be used for all of theconstructs in the figure, although the kappa constant light chain canalso be substituted.

It should be noted that these bottle opener backbones find use in theCentral-scFv format of FIG. 1F, where an additional, second Fab (vh-CH1and vl-constant light) with the same antigen binding as the first Fab isadded to the N-terminus of the scFv on the “bottle opener side”.

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIG. 10A to 10E depicts the sequences for a select number of anti-PD-1antibodies. It is important to note that these sequences were generatedbased on human IgG1, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_/S267K”) which is depictedin FIG. 6A. The CDRs are underlined. As noted herein and is true forevery sequence herein containing CDRs, the exact identification of theCDR locations may be slightly different depending on the numbering usedas is shown in Table 1, and thus included herein are not only the CDRsthat are underlined but also CDRs included within the VH and VL domainsusing other numbering systems.

FIG. 11A to 11E depict a select number of PD-1 ABDs, with additionalanti-PD-1 ABDs being listed as SEQ 1-2392, 3125-3144, 4697-7594 and4697-21810. The CDRs are underlined, the scFv linker is doubleunderlines (in the sequences, the scFv linker is a positively chargedscFv (GKPGS)4 linker (SEQ ID NO: 26209), although as will be appreciatedby those in the art, this linker can be replaced by other linkers,including uncharged or negatively charged linkers, some of which aredepicted in FIG. 8), and the slashes indicate the border(s) of thevariable domains. In addition, the naming convention illustrates theorientation of the scFv from N- to C-terminus; some of the sequences inthis Figure are oriented as VH-scFv linker-VL (from N- to C-terminus),while some are oriented as VL-scFv linker-VH (from N- to C-terminus),although as will be appreciated by those in the art, these sequences mayalso be used in the opposition orientation from their depiction herein.As noted herein and is true for every sequence herein containing CDRs,the exact identification of the CDR locations may be slightly differentdepending on the numbering used as is shown in Table 1, and thusincluded herein are not only the CDRs that are underlined but also CDRsincluded within the VH and VL domains using other numbering systems.Furthermore, as for all the sequences in the Figures, these VH and VLsequences can be used either in a scFv format or in a Fab format.

FIG. 12A to 12PP depict a select number of CTLA-4 ABDs, with additionalanti-CTLA-4 ABDs being listed as SEQ ID NO:2393-2414 and 3737-3816. TheCDRs are underlined, the scFv linker is double underlines (in thesequences, the scFv linker is a positively charged scFv (GKPGS)4 linker(SEQ ID NO: 26209), although as will be appreciated by those in the art,this linker can be replaced by other linkers, including uncharged ornegatively charged linkers, some of which are depicted in FIG. 8), andthe slashes indicate the border(s) of the variable domains. In addition,the naming convention illustrates the orientation of the scFv from N- toC-terminus; some of the sequences in this Figure are oriented as VH-scFvlinker-VL (from N- to C-terminus), while some are oriented as VL-scFvlinker-VH (from N- to C-terminus), although as will be appreciated bythose in the art, these sequences may also be used in the oppositionorientation from their depiction herein. As noted herein and is true forevery sequence herein containing CDRs, the exact identification of theCDR locations may be slightly different depending on the numbering usedas is shown in Table 1, and thus included herein are not only the CDRsthat are underlined but also CDRs included within the VH and VL domainsusing other numbering systems. Furthermore, as for all the sequences inthe Figures, these VH and VL sequences can be used either in a scFvformat or in a Fab format.

FIG. 13A to 13N depict a select number of LAG-3 ABDs, with additionalanti-LAG-3 ABDs being listed as SEQ ID NO:2415-2604 and 3817-3960. TheCDRs are underlined, the scFv linker is double underlines (in thesequences, the scFv linker is a positively charged scFv (GKPGS)4 linker(SEQ ID NO: 26209), although as will be appreciated by those in the art,this linker can be replaced by other linkers, including uncharged ornegatively charged linkers, some of which are depicted in FIG. 8), andthe slashes indicate the border(s) of the variable domains> In addition,the naming convention illustrates the orientation of the scFv from N- toC-terminus; some of the sequences in this Figure are oriented as VH-scFvlinker-VL (from N- to C-terminus), while some are oriented as VL-scFvlinker-VH (from N- to C-terminus), although as will be appreciated bythose in the art, these sequences may also be used in the oppositionorientation from their depiction herein. As noted herein and is true forevery sequence herein containing CDRs, the exact identification of theCDR locations may be slightly different depending on the numbering usedas is shown in Table 1, and thus included herein are not only the CDRsthat are underlined but also CDRs included within the VH and VL domainsusing other numbering systems. Furthermore, as for all the sequences inthe Figures, these VH and VL sequences can be used either in a scFvformat or in a Fab format.

FIG. 14A to 14I depicts the sequences for a select number of anti-TIM-3antibodies. It is important to note that these sequences were generatedbased on human IgG1 backbone, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_/S267K”) although otherformats can be used as well. The CDRs are underlined. As noted hereinand is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table 1, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems.

FIG. 15A to 15C depicts the sequences for a select number of anti-PD-L1antibodies. It is important to note that these sequences were generatedbased on human IgG1 backbone, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_/S267K”) as outlined herein,although other formats can be used as well. The CDRs are underlined. Asnoted herein and is true for every sequence herein containing CDRs, theexact identification of the CDR locations may be slightly differentdepending on the numbering used as is shown in Table 1, and thusincluded herein are not only the CDRs that are underlined but also CDRsincluded within the VH and VL domains using other numbering systems.

FIG. 16 depicts the sequences for a prototype anti-4-1BB antibody. It isimportant to note that these sequences were generated based on humanIgG1 backbone, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_/S267K”), although the otherformats can be used as well. The CDRs are underlined. As noted hereinand is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table 1, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems.

FIG. 17 depicts the sequences for a prototype anti-OX40 antibody. It isimportant to note that these sequences were generated based on humanIgG1 backbone, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_/S267K”), although otherformats can be used as well. The CDRs are underlined. As noted hereinand is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table 1, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems.

FIG. 18 depicts the sequences for a prototype anti-GITR antibody. It isimportant to note that these sequences were generated based on humanIgG1 backbone, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_/S267K”), although otherformats can be used as well. The CDRs are underlined. As noted hereinand is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table 1, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems.

FIGS. 19A to 19G depicts the sequences for prototype anti-ICOSantibodies. It is important to note that these sequences were generatedbased on human IgG1 backbone, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_/S267K”), although otherformats can be used as well. The CDRs are underlined. As noted hereinand is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table X, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems.

FIGS. 20A to 20G depicts sequences for exemplary anti-ICOS Fabs. TheCDRs are underlined and slashes (/) indicate the border(s) of thevariable regions. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on the numbering used as is shown in TableX, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the VH and VL domains using othernumbering systems. Furthermore, as for all the sequences in the Figures,these VH and VL sequences can be used either in a scFv format or in aFab format. It is important to note that these sequences were generatedusing six-histidine (His6 or HHHHHH) (SEQ ID NO: 28666) C-terminal tagsat the C-terminus of the heavy chains, which have been removed.

FIGS. 21A to 21B depicts melting temperatures (Tm) and changes inmelting temperature from the parental Fab (XENP22050) as determined byDSF of variant anti-ICOS Fabs engineered for stability.

FIGS. 22A to 22C depicts equilibrium dissociation constants (KD),association rates (ka), and dissociation rates (kd) of variant anti-ICOSFabs for murine Fc fusions of human ICOS captured on AMC biosensors asdetermined by Octet.

FIG. 23 depicts equilibrium dissociation constants (KD), associationrates (ka), and dissociation rates (kd) of variant anti-ICOS Fabs forbiotinylated IgG1 Fc fusions of human ICOS captured on SA biosensors asdetermined by Octet.

FIGS. 24A to 24M depicts sequences for exemplary anti-ICOS scFvs. TheCDRs are underlined, the scFv linker is double underline (in thesequences, the scFv linker is a positively charged scFv (GKPGS)4 linker(SEQ ID NO: 26209), although as will be appreciated by those in the art,this linker can be replaced by other linkers, including uncharged ornegatively charged linkers, some of which are depicted in Figure X), andslashes (/) indicate the border(s) of the variable regions. The namingconvention illustrates the orientation of the scFv from N- toC-terminus; some of the sequences in this Figure are oriented as VH-scFvlinker-VL (from N- to C-terminus, see FIG. 24), while some are orientedas VL-scFv linker-VH (from N- to C-terminus, see FIG. 24B), although aswill be appreciated by those in the art, these sequences may also beused in the opposition orientation from their depiction herein. As notedherein and is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table X, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems. Furthermore, as forall the sequences in the Figures, these VH and VL sequences can be usedeither in a scFv format or in a Fab format. It is important to note thatthese sequences were generated using polyhistidine (His6 or HHHHHH) (SEQID NO: 28666) C-terminal tags at the C-terminus of the heavy chains,which have been removed.

FIG. 25 depicts melting temperatures (Tm) and changes in meltingtemperature from the parental scFv (XENP24352; oriented as VH-scFvlinker-VL from N- to C-terminus) as determined by DSF of variantanti-ICOS scFvs engineered for stability.

FIG. 26A to 26D depicts the amino acid sequences of prototypeanti-costim×anti-checkpoint antibodies in the bottle-opener format(Fab-scFv-Fc). The antibodies are named using the Fab variable regionfirst and the scFv variable region second, separated by a dash, followedby the chain designation (Fab-Fc heavy chain, scFv-Fc heavy chain orlight chain). CDRs are underlined and slashes indicate the border(s) ofthe variable regions. The scFv domain has different orientations (N- toC-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH asindicated, although this can be reversed. In addition, each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIG. 27 depicts induction of cytokine secretion by prototypecostim/checkpoint bottle-openers in an SEB-stimulated PBMC assay.

FIG. 28 depicts induction of IL-2 secretion in naive (non-SEBstimulated) and SEB-stimulated PBMCs following treatment with theindicated test articles.

FIG. 29 depicts a schematic associated with the benefit of acostim×checkpoint blockade bispecific antibody, showing that becausetumor TILs co-express immune checkpoint receptors and costimulatoryreceptors, a bispecific antibody increases specificity, enhancinganti-tumor activity and avoiding peripheral toxicity.

FIG. 30 depicts that double-positive cells are selectively occupied byexemplary anti-ICOS×anti-PD-1 antibody (XENP20896) as compared toone-arm anti-PD-1 antibody (XENP20111) and one-arm anti-ICOS antibody(XENP20266).

FIG. 31A to 31B shows the receptor occupancy of anti-ICOS×anti-PD-1bispecific antibody (XENP20896), one-arm anti-ICOS antibody (XENP20266)and one-arm anti-PD-1 antibody (XENP20111) on A) PD-1 and ICOSdouble-positive T cells and B) PD-1 and ICOS double-negative T cellsafter SEB stimulation of human PBMCs.

FIG. 32A to 32D depicts the amino acid sequences of exemplaryanti-ICOS×anti-PD-1 antibodies in the bottle-opener format(Fab-scFv-Fc). The antibodies are named using the Fab variable regionfirst and the scFv variable region second, separated by a dash, followedby the chain designation (Fab-Fc heavy chain, scFv-Fc heavy chain orlight chain). CDRs are underlined and slashes indicate the border(s) ofthe variable regions. The scFv domain has different orientations (N- toC-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH asindicated, although this can be reversed. In addition, each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIG. 33A to 33C depicts the amino acid sequences of exemplaryanti-ICOS×anti-PD-1 antibodies in the bottle-opener format (Fab-scFv-Fc)which include the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum. The antibodies arenamed using the Fab variable region first and the scFv variable regionsecond, separated by a dash, followed by the chain designation (Fab-Fcheavy chain, scFv-Fc heavy chain or light chain). CDRs are underlinedand slashes indicate the border(s) of the variable regions. The scFvdomain has different orientations (N- to C-terminus) of either VH-scFvlinker-VL or VL-scFv linker-VH as indicated, although this can bereversed.

FIG. 34A to 34C depicts equilibrium dissociation constants (KD),association rates (ka), and dissociation rates (kd) of variantanti-ICOS×anti-PD-1 bispecific antibodies for murine Fc fusion of humanICOS captured on AMC biosensors as determined by Octet.

FIG. 35 depicts equilibrium dissociation constants (KD), associationrates (ka), and dissociation rates (kd) of variant anti-ICOS×anti-PD-1bispecific antibodies for biotinylated IgG1 Fc fusions of human and ICOScaptured on SA/SAX biosensors as determined by Octet.

FIGS. 36A and 36B depicts cell surface binding of variantanti-ICOS×anti-PD-1 bispecific to human T cells in SEB-stimulated PBMCassays in two separate experiments depicted in A) and B).

FIG. 37 shows the receptor occupancy of variant anti-ICOS×anti-PD-1bispecific antibodies, one-arm anti-ICOS antibodies and one-armanti-PD-1 antibody (XENP20111) on PD-1 and ICOS double-positive T cellsafter SEB stimulation of human PBMCs.

FIGS. 38A and 38B show that variant anti-ICOS×anti-PD-1 bispecificantibodies promote A) IL-2 and B) IFNγ secretion from SEB stimulatedPBMCs.

FIGS. 39A and 39B show that variant anti-ICOS×anti-PD-1 bispecificantibodies promote A) IL-2 and B) IFNγ secretion from SEB-stimulatedPBMCs.

FIGS. 40A and 40B depicts the concentration of IFNγ in mice on Day A) 7and B) 11 after engraftment with human PBMCs and treatment with theindicated test articles.

FIGS. 41A and 41B depicts CD45+ cell counts in mice as determined byflow cytometry on Day A) 11 and B)14 after engraftment with human PBMCsand treatment with the indicated test articles.

FIGS. 42A and 42B depicts A) CD8+ T cell and B) CD4+ T cell counts inmice as determined by flow cytometry on Day 14 after engraftment withhuman PBMCs and treatment with the indicated test articles (**p≤0.01).

FIG. 43 depicts the change in body weight in mice by Day 14 afterengraftment with human PBMCs and treatment with the indicated testarticles (**p≤0.01).

FIGS. 44A and 44B depicts the concentration of IFNγ in mice on Day A) 7and B) 14 after engraftment with human PBMCs and treatment with theindicated test articles.

FIG. 45 depicts CD45+ cell counts in mice as determined by flowcytometry on Day 14 after engraftment with human PBMCs and treatmentwith the indicated test articles.

FIG. 46A to 46C depicts A) CD8+ T cell and B) CD4+ T cell counts in miceas determined by flow cytometry on Day 14 after engraftment with humanPBMCs and treatment with the indicated test articles.”

FIGS. 47A and 47B depicts the change in body weight in mice by Day 12and 15 after engraftment with human PBMCs and treatment with theindicated test articles.

FIG. 48A to 48D depicts the amino acid sequences of exemplaryanti-ICOS×anti-PD-1 antibodies in the bottle-opener format (Fab-scFv-Fc)with alternative ICOS ABDs. The antibodies are named using the Fabvariable region first and the scFv variable region second, separated bya dash, followed by the chain designation (Fab-Fc heavy chain, scFv-Fcheavy chain or light chain). CDRs are underlined and slashes indicatethe border(s) of the variable regions. The scFv domain has differentorientations (N- to C-terminus) of either VH-scFv linker-VL or VL-scFvlinker-VH as indicated, although this can be reversed. In addition, eachsequence outlined herein can include or exclude the M428L/N434S variantsin one or preferably both Fc domains, which results in longer half-lifein serum.

FIG. 49 depict cytokine release assay for IL-2 after SEB-stimulation ofhuman PBMCs and treatment with alternative anti-ICOS×anti-PD-1bispecific antibodies.

FIG. 50 depict cytokine release assay for IL-2 (as fold induction overbivalent anti-RSV mAb) after SEB-stimulation of human PBMCs andtreatment with alternative anti-ICOS×anti-PD-1 bispecific antibodies.

FIG. 51 depicts AKT phosphorylation in SEB-stimulated purified CD3+ Tcells after treatment with anti-ICOS×anti-PD-1 bispecific antibodies.

FIG. 52 depicts fold induction of A) IL-17A, B) IL17F, C) IL-22, D)IL-10, E) IL-9, and F) IFNγ gene expression by the indicated testarticles over induction by bivalent anti-RSV as determined byNanoString.

FIG. 53A to 53F depict mean fold induction in expression of selectedimmune response genes by indicated test articles over treatment withbivalent anti-RSV mAb as determined by NanoString. The shading intensitycorresponds to the magnitude of the fold change.

FIG. 54 depicts CD45+ cell counts in mice as determined by flowcytometry on Day 14 after engraftment with human PBMCs and treatmentwith the indicated test articles.

FIGS. 55A to 55D, similar to FIG. 9 and FIG. 75, depicts the sequencesof the “backbone” portion (e.g. without the Fvs) of a number ofadditional formats, including the Central scFv of FIG. 2F, theCentral-scFv2 format of FIG. 2J, the bispecific mAb of FIG. 2K, theDVD-Ig of FIG. 2L and the Trident format of FIG. 2M. In FIG. 2L, theDVD-Ig® linkers are shown with double underlining, with other linkersfound in WO2007/024715, hereby incorporated by reference in its entiretyand in particular for those sequences. In the Trident format, otherTrident linkers and coil-coil sequences are shown in WO 2015/184203,hereby incorporated by reference in its entirety and in particular forthose sequences. As will be appreciated by those in the art, boldeddomains (e.g. “VH1”, VH2-scFv linker-VL2″, etc.) are separated withslashes “/”, and may include optional domain linkers as needed. All ofthese backbones utilize the kappa constant region for the light chain,although the lambda chain can also be used. As for FIG. 9 and FIG. 75,these backbones can be combined with any vh and vl domains as outlinedherein.

FIGS. 56A and 56B depicts the amino acid sequence of illustrativeanti-PD-1×anti-ICOS antibodies in the bottle-opener format(Fab-scFv-Fc). The antibodies are named using the Fab variable regionfirst and the scFv variable region second, separated by a dash, followedby the chain designation (Fab-Fc heavy chain, scFv-Fc heavy chain orlight chain). CDRs are underlined and slashes indicate the border(s) ofthe variable regions. The scFv domain has different orientations (N- toC-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH asindicated, although this can be reversed. In addition, each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIG. 57A to 57C depicts the amino acid sequence of illustrativeanti-PD-1×anti-ICOS antibodies in the central-scFv format. Theantibodies are named using the first Fab-Fc variable region first andthe Fab-scFv-Fc variable region second, separated by a dash, followed bythe chain designation (Fab-Fc heavy chain, Fab-scFv-Fc heavy chain orlight chain). CDRs are underlined and slashes indicate the border(s) ofthe variable regions. The scFv domain has different orientations (N- toC-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH asindicated, although this can be reversed. In addition, each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIG. 58 depicts the amino acid sequence of illustrativeanti-PD-1×anti-ICOS antibodies in the central-scFv2 format. Theantibodies are named using the Fab variable region first and the scFvvariable region second, followed by the chain designation (heavy chainor light chain). CDRs are underlined and slashes indicate the border(s)of the variable regions. The scFv domain has different orientations (N-to C-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH asindicated, although this can be reversed. In addition, each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIG. 59 depicts the amino acid sequence of an illustrativeanti-PD-1×anti-ICOS antibody in the bispecific mAb format. Theantibodies are named using the first Fab variable region for a firstantigen and the second Fab variable region for a second antigen,separated by a dash, followed by the chain designation (Heavy Chain 1 orLight Chain 1 for the first antigen and Heavy Chain 2 or Light Chain 2for the second antigen). CDRs are underlined and slashes indicate theborder(s) of the variable regions. Each sequence outlined herein caninclude or exclude the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum.

FIG. 60 depicts the amino acid sequence of an illustrativeanti-PD-1×anti-ICOS antibody in the DVD-IgG format. The antibodies arenamed using the first variable region for a first antigen and the secondFab variable region for a second antigen, followed by the chaindesignation (Heavy Chain or Light Chain). CDRs are underlined andslashes indicate the border(s) of the variable regions. Each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIGS. 61A and 61B depicts the amino acid sequence of an illustrativeanti-PD-1×anti-ICOS antibody in the Trident format. The antibodies arenamed using the VL and VH of a first antigen which comprises a DART andthe Fab variable region for a second antigen, separated by a dash,followed by the chain designation (Heavy Chain or Light Chain). CDRs areunderlined and slashes indicate the border(s) of the variable regions.Each sequence outlined herein can include or exclude the M428L/N434Svariants in one or preferably both Fc domains, which results in longerhalf-life in serum.

FIG. 62 depicts induction of cytokine secretion (IL-2) by alternativeformat costim×checkpoint blockade bispecific antibodies in anSEB-stimulated PBMC assay.

FIG. 63 depicts the amino acid sequences of an illustrativeanti-ICOS×anti-CTLA-4 antibody in the bottle-opener format(Fab-scFv-Fc). The antibodies are named using the Fab variable regionfirst and the scFv variable region second, separated by a dash, followedby the chain designation (Fab-Fc heavy chain, scFv-Fc heavy chain orlight chain). CDRs are underlined and slashes indicate the border(s) ofthe variable regions. The scFv domain has different orientations (N- toC-terminus) of either VH-scFv linker-VL or VL-scFv linker-VH asindicated, although this can be reversed. In addition, each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIGS. 64A and 64B depicts the amino acid sequence of illustrativeanti-LAG-3×anti-ICOS antibodies in the bispecific mAb format. Theantibodies are named using the first Fab variable region for a firstantigen and the second Fab variable region for a second antigen,separated by a dash, followed by the chain designation (Heavy Chain 1 orLight Chain 1 for the first antigen and Heavy Chain 2 or Light Chain 2for the second antigen). CDRs are underlined and slashes indicate theborder(s) of the variable regions. Each sequence outlined herein caninclude or exclude the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum.

FIG. 65 depicts the amino acid sequence of an illustrativeanti-TIM-3×anti-ICOS antibody in the bispecific mAb format. Theantibodies are named using the first Fab variable region for a firstantigen and the second Fab variable region for a second antigen,separated by a dash, followed by the chain designation (Heavy Chain 1 orLight Chain 1 for the first antigen and Heavy Chain 2 or Light Chain 2for the second antigen). CDRs are underlined and slashes indicate theborder(s) of the variable regions. Each sequence outlined herein caninclude or exclude the M428L/N434S variants in one or preferably both Fcdomains, which results in longer half-life in serum.

FIG. 66A to 66C depicts the amino acid sequences of anti-ICOS×anti-PD-L1antibodies in the bottle-opener format (Fab-scFv-Fc) and central-scFv2format. The bottle-openers are named using the Fab variable region firstand the scFv variable region second, separated by a dash, followed bythe chain designation (Fab-Fc heavy chain, scFv-Fc heavy chain or lightchain). Central-scFv2s are named using the Fab variable region first andthe scFv variable region second, followed by the chain designation(heavy chain or light chain). CDRs are underlined and slashes indicatethe border(s) of the variable regions. CDRs are underlined and slashesindicate the border(s) of the variable regions. The scFv domain hasdifferent orientations (N- to C-terminus) of either VH-scFv linker-VL orVL-scFv linker-VH as indicated, although this can be reversed. Inaddition, each sequence outlined herein can include or exclude theM428L/N434S variants in one or preferably both Fc domains, which resultsin longer half-life in serum.

FIG. 67 depicts induction of cytokine secretion (IL-2) by additionalcostim×checkpoint blockade bispecific antibodies in an SEB-stimulatedPBMC assay.

FIGS. 68A to 68G depict amino acid sequences for exemplary one-armanti-ICOS Fab-Fc antibodies. CDRs are underlined and slashes indicatethe border(s) of variable regions. These are referred to as “one-arm” or“one armed” formats as one amino acid chain is only an Fc domain, withthe other side being an anti-ICOS Fab side. The Fc domain contains theS364K/E357Q skew variants, as well as the pI(−) Isosteric A variantsdepicted in Figure X. The Fab Fc domain contains the L368D/K370S skewvariants as well as the pI ISO(+RR) variants depicted in Figure X. BothFc domains include the ablation variants(E233P/L234V/L235A/G236del/S267K).

FIG. 69 depicts equilibrium dissociation constants (KD), associationrates (ka), and dissociation rates (kd) of variant one-arm anti-ICOSFab-Fc antibodies for murine Fc fusions of human ICOS captured on AMCbiosensors as determined by Octet.

FIG. 70 depicts AKT phosphorylation in SEB-stimulated purified CD3+ Tcells after treatment with bivalent and monovalent anti-PD-1 antibodiesand anti-ICOS×anti-PD-1 bispecific antibodies.

FIG. 71 depicts AKT phosphorylation in purified CD3+ T cells aftertreatment with monovalent anti-ICOS Fab-Fc antibodies with alternativeanti-ICOS ABDs.

FIG. 72A to 72C depict some prototype bispecific antibodies (OX40×PD-1,GITR×PD-1, 4-1BB×PD-1, CTLA-4×ICOS).

FIG. 73A to 73H depict some prototype mAbs (4-1BB, OX40, GITR, ICOS,PD-L1 and PD-1), the Fvs of which can be used in combination with theother Fvs of the invention and in any format (bottle opener, mAb-Fv,mAb-scFv, central-scFv, bispecific mAb, central-Fv, one armedcentral-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or Trident). Someadditional ICOS X PD-L1 bottle opener sequences are shown as well.

FIG. 74A to 74F depict additional PD-1×ICOS bottle openers, in somecases with the PD-1 Fv being in the Fab format and the ICOS Fv in a scFvformat and in other cases reversed.

FIG. 75A to 75D shows the sequences of a mAb-scFv backbone of use in theinvention, to which the Fv sequences of the invention are added.mAb-scFv backbone 1 is based on human IgG1 (356E/358M allotype), andincludes the S364K/E357Q: L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 2 is based on human IgG1 (356D/358L allotype), and includes theS364K/E357Q: L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 3 is based on human IgG1 (356E/358M allotype), and includes theS364K/E357Q: L368D/K370S skew variants, N208D/Q295E/N384D/Q418E/N421D pIvariants on the Fab side and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains, as well as an N297A variant on bothchains. Backbone 4 is identical to 3 except the mutation is N297S.Alternative formats for mAb-scFv backbones 3 and 4 can exclude theablation variants E233P/L234V/L235A/G236del/S267K in both chains.Backbone 5 is based on human IgG4, and includes the S364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421D pI variantson the Fab side and the E233P/L234V/L235A/G236del/S267K ablationvariants on both chains, as well as a S228P (EU numbering, this is S241Pin Kabat) variant on both chains that ablates Fab arm exchange as isknown in the art Backbone 6 is based on human IgG2, and includes theS364K/E357Q: L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the Fab side. Backbone 7 isbased on human IgG2, and includes the S364K/E357Q: L368D/K370S skewvariants, the N208D/Q295E/N384D/Q418E/N421D pI variants on the Fab sideas well as a S267K variant on both chains.

As will be appreciated by those in the art and outlined below, thesesequences can be used with any vh and vl pairs outlined herein, with onemonomer including both a Fab and an scFv (optionally including a chargedscFv linker) and the other monomer including the Fab sequence (e.g. a vhattached to the “Fab side heavy chain” and a vl attached to the“constant light chain”). That is, any Fv sequences outlined herein foranti-CTLA-4, anti-PD-1, anti-LAG-3, anti-TIM-3, anti-TIGIT, anti-BTLA,anti-ICOS, anti-GITR, anti-OX40 and anti-4-1BB, whether as scFv (again,optionally with charged scFv linkers) or as Fabs, can be incorporatedinto this FIG. 75 backbone in any combination. The monomer 1 side is theFab-scFv pI negative side, and includes the heterodimerization variantsL368D/K370S, the isosteric pI variants N208D/Q295E/N384D/Q418E/N421D,the ablation variants E233P/L234V/L235A/G236del/S267K, (all relative toIgG1). The monomer 2 side is the scFv pI positive side, and includes theheterodimerization variants 364K/E357Q. However, other skew variantpairs can be substituted, particularly [S364K/E357Q: L368D/K370S];[L368D/K370S: S364K]; [L368E/K370S: S364K]; [T411T/E360E/Q362E: D401K];[L368D/K370S: S364K/E357L], [K370S: S364K/E357Q], [T366S/L368A/Y407V:T366W] and [T366S/L368A/Y407V/Y394C: T366W/S354C].

The constant light chain depicted in FIG. 75A can be used for all of theconstructs in the figure, although the kappa constant light chain canalso be substituted.

It should be noted that these mAb-scFv backbones find use in the boththe mAb-Fv format of FIG. 1H (where one monomer comprises a vl at theC-terminus and the other a vh at the C-terminus) as well as the scFv-mAbformat (with a scFv domain added to the C-terminus of one of themonomers).

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIG. 76A to 76F depict a number of prior art sequences for Fvs that bindhuman PD-1 as vh and vl sequences. As will be appreciated by those inthe art, any of these Fvs can be combined with an Fv that binds acostimulatory receptor (e.g. ICOS, GITR, OX40 or 4-1BB, including the Fvsequences contained herein) and in any format (bottle opener, mAb-Fv,mAb-scFv, central-scFv, bispecific mAb, central-Fv, one armedcentral-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or Trident). Inparticular they can be combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.

FIGS. 77A and 77B depict a number of prior art sequences for Fvs thatbind human ICOS as vh and vl sequences. As will be appreciated by thosein the art, any of these Fvs can be combined with an Fv that binds acheckpoint receptor (e.g. PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, TIGIT andBTLA, including the Fv sequences contained herein) and in any format(bottle opener, mAb-Fv, mAb-scFv, central-scFv, bispecific mAb,central-Fv, one armed central-scFv, one armed scFv-mAb, dual scFv,DVD-Ig or Trident). In particular they can be combined with PD-1 ABDshaving the identifiers 1G6_H1.279_L1.194; 1G6_H1.280_L1.224;1G6_L1.194_H1.279; 1G6_L1.210_H1.288; and 2E9_H1L1.

FIG. 78A to 78J depict a number of prior art sequences for Fvs that bindhuman PD-L1 as vh and vl sequences. As will be appreciated by those inthe art, any of these Fvs can be combined with an Fv that binds acostimulatory receptor (e.g. ICOS, GITR, OX40 or 4-1BB, including the Fvsequences contained herein) and in any format (bottle opener, mAb-Fv,mAb-scFv, central-scFv, bispecific mAb, central-Fv, one armedcentral-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or Trident). Inparticular they can be combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.

FIGS. 79A and 79B depict a number of prior art sequences for Fvs thatbind human CTLA-4 as vh and vl sequences. As will be appreciated bythose in the art, any of these Fvs can be combined with an Fv that bindsa costimulatory receptor (e.g. ICOS, GITR, OX40 or 4-1BB, including theFv sequences contained herein) and in any format (bottle opener, mAb-Fv,mAb-scFv, central-scFv, bispecific mAb, central-Fv, one armedcentral-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or Trident). Inparticular they can be combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.

FIG. 80A to 80C depict a number of prior art sequences for Fvs that bindhuman LAG-3 as vh and vl sequences. As will be appreciated by those inthe art, any of these Fvs can be combined with an Fv that binds acostimulatory receptor (e.g. ICOS, GITR, OX40 or 4-1BB, including the Fvsequences contained herein) and in any format (bottle opener, mAb-Fv,mAb-scFv, central-scFv, bispecific mAb, central-Fv, one armedcentral-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or Trident). Inparticular they can be combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.

FIG. 81A to 81C depict a number of prior art sequences for Fvs that bindhuman TIM-3 as vh and vl sequences. As will be appreciated by those inthe art, any of these Fvs can be combined with an Fv that binds acostimulatory receptor (e.g. ICOS, GITR, OX40 or 4-1BB, including the Fvsequences contained herein) and in any format (bottle opener, mAb-Fv,mAb-scFv, central-scFv, bispecific mAb, central-Fv, one armedcentral-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or Trident). Inparticular they can be combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.

FIG. 82 depict a number of prior art sequences for Fvs that bind humanBTLA as vh and vl sequences. As will be appreciated by those in the art,any of these Fvs can be combined with an Fv that binds a costimulatoryreceptor (e.g. ICOS, GITR, OX40 or 4-1BB, including the Fv sequencescontained herein) and in any format (bottle opener, mAb-Fv, mAb-scFv,central-scFv, bispecific mAb, central-Fv, one armed central-scFv, onearmed scFv-mAb, dual scFv, DVD-Ig or Trident). In particular they can becombined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.

FIG. 83A to 83D depict a number of prior art sequences for Fvs that bindhuman TIGIT as vh and vl sequences. As will be appreciated by those inthe art, any of these Fvs can be combined with an Fv that binds acostimulatory receptor (e.g. ICOS, GITR, OX40 or 4-1BB, including the Fvsequences contained herein) and in any format (bottle opener, mAb-Fv,mAb-scFv, central-scFv, bispecific mAb, central-Fv, one armedcentral-scFv, one armed scFv-mAb, dual scFv, DVD-Ig or Trident). Inparticular they can be combined with ICOS]_H0L0 and [ICOS]H0.66_L0 ABDs.

FIG. 84A to 84C depict a number of BTLA ABDs, with additional anti-BTLAABDs being listed as SEQ ID NO: 3705-3736. The CDRs are underlined, thescFv linker is double underlined (in the sequences, the scFv linker is apositively charged scFv (GKPGS)4 linker (SEQ ID NO: 26209), although aswill be appreciated by those in the art, this linker can be replaced byother linkers, including uncharged or negatively charged linkers, someof which are depicted in FIG. 8), and the slashes indicate the border(s)of the variable domains. As above, the naming convention illustrates theorientation of the scFv from N- to C-terminus; in the sequences listedin this figure, they are all oriented as vh-scFv linker-vl (from N- toC-terminus), although these sequences may also be used in the oppositeorientation, (from N- to C-terminus) vl-linker-vh. As noted herein andis true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table 1, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe vh and vl domains using other numbering systems. Furthermore, as forall the sequences in the Figures, these vh and vl sequences can be usedeither in a scFv format or in a Fab format.

FIG. 85 is a matrix of possible combinations of the costim andcheckpoint ABDs, with all possible combinations possible. An “A” in thebox means that the PD-1 ABD is 1G6_L1.194_H1.279. A “B” in the box meansthat the ICOS ABD is [ICOS]H.066_L0. A “C” in the box means that thePD-1 is the scFv in the pair. A “D” in the box means the CTLA-4 ABD is aFab and is [CTLA-4]_H3 L0.22. An “E” in the box means that the CTLA-4ABD is a scFv and is [CTLA-4]_H3.23_L0.22. An “F” in the box means thatthe LAG-3 ABD is 7G8_H3.30_L1.34. A “G” in the box means that the BTLAABD is 9C6_H1.1_L1. An “H” in the box means that combination is a bottleopener. An “I” in the box means the combination is Central-scFv format.

FIG. 86 depicts two more ICOS X PD-1 bottle openers.

FIGS. 87A and 87B depicts that monovalent engagement of PD-1 and ICOSenables avid targeting of double positive T cells; receptor occupancy ofPD1 and ICOS on human CD3+T cells stimulated with SEB. Scatterplots areshown for 3 ug/mL concentration.

FIG. 88 depicts that XmAb23104 exhibits a multi-gene expressionsignature consistent with ICOS co-stimulation. SEB-stimulated humanPBMCs (multiple healthy donors). RNA analyzed on a NanoString platformas described herein using the nCountrer PanCancer Immune ProfilingPanel.

FIG. 89 shows the PD1×CTLA4 bispecific antibody is highly active in amouse model for checkpoint blockade.

FIG. 90 depicts binding of the indicated test articles to SEB-stimulatedCD3+ T cells from 3 separate PBMC donors (A-C).

FIG. 91 depicts checkpoint receptor occupancy by the indicated testarticles as indicated by percentage of populations of SEB-stimulatedCD3+ T cells with unoccupied PD-1 and/or ICOS receptors as shown bystaining.

FIG. 92 shows the amount of unoccupied A) PD-1 and B) ICOS receptors onCD3+ICOS+PD-1+ SEB-stimulated T cells following treatment with theindicated test articles.

FIG. 93 depicts fold change (x-axis) in expression of immune-relatedgenes by SEB-stimulated T cells following treatment with A) XmAb23104,B) anti-PD-1 mAb (XENP19686), C) anti-ICOS mAb (XENP16435), and D)anti-PD-1 mAb (XENP19686) in combination with anti-ICOS mAb (XENP16435)over treatment with negative control anti-RSV mAb (XENP15074).

FIG. 94 depicts tumor burden (as indicated by mean tumor volume asdetermined by caliper measurements) in NSG mice engrafted withpp65-expressing MCF-7 cells followed by engraftment with huPBMCsfollowing treatment with XmAb23104. Triangles below the X-axis indicatedosing days.

FIG. 95 depicts tumor burden (as indicated by mean tumor volume asdetermined by caliper measurements) in NSG mice engrafted withpp65-expressing MDA-MB-231 cells followed by engraftment with huPBMCsfollowing treatment with XmAb23104. Triangles below the X-axis indicatedosing days.

FIG. 96 depicts the amino acid sequences of a monovalent anti-ICOSFab-Fc fusion based on the anti-ICOS arm from XmAb23104 with M428L/N434SXtend Fc. CDRs are underlined and slashes indicate the border(s) of thevariable regions.

FIG. 97 depicts the sequences for A) human PD-L1 and B) human PD-L2fused to murine IgG2aa Fc region. Slashes (/) indicate the borderbetween the PD-1 ligand and the murine IgG2aa Fc-region.

FIGS. 98A and B depicts binding of A) PD-L1 and B) PD-L2 to PD-1+ICOS+cells following treatment with the indicated test articles.

FIG. 99 depicts the sequence for human ICOSL fused to human IgG1 Fcregion with E233P/L234V/L235A/G236del/S267K substitutions. Slashes (/)indicate the border between the PD-1 ligand and the human IgG1Fc-region.

FIG. 100 depicts binding of ICOSL to PD-1+ICOS+ cells followingtreatment with the indicated test articles.

FIG. 101A to 101D depicts binding of XmAb23104 to CD3+ T cells inSEB-stimulated PBMCs from 4 cynomolgus monkeys.

FIGS. 102A and 102B depicts fold induction of A) IFNγ and B) IL-2 overbivalent anti-RSV mAb by the indicated test articles in anSEB-stimulated PBMC assay.

FIG. 103 depicts IL-2 induction by XENP15074, XENP16432, and XmAb23104in an SEB-stimulated PBMC assay.

FIG. 104 depicts the amino acid sequences of a monovalent anti-PD-1scFv-Fc fusion based on the anti-PD-1 arm from XmAb23104 withM428L/N424S Xtend Fc. CDRs are underlined and slashes indicate theborder(s) of the variable regions.

FIG. 105 depicts fold induction of IFNγ over bivalent anti-RSV mAb bythe indicated test articles in an MLR assay.

FIGS. 106A and 106B depicts IFNγ induction by XmAb23104 or a combinationof XENP20111 and XENP24901 (at equimolar PD-1 and ICOS bindingconcentrations relative to XmAb23104) in SEB-stimulated PBMCs from 2separate donors.

FIGS. 107A and 107B depicts IL-2 induction by XmAb23104 or a combinationof XENP20111 and XENP24901 (at equimolar PD-1 and ICOS bindingconcentrations relative to XmAb23104) in SEB-stimulated PBMCs from 2separate donors.

FIG. 108 depicts the amino acid sequences of a bivalent anti-CTLA-4 mAbbased on ipilimumab and human IgG1 Fc region withE233P/L234V/L235A/G236del/S267K substitutions. CDRs are underlined andslashes indicate the border(s) of the variable regions.

FIGS. 109A and 109B depict fold induction of A) IFNγ and B) IL-2 overbivalent anti-RSV mAb by XmAb23104 alone, XENP16433 (anti-CTLA-4 mAbbased on ipilimumab), or XmAb23104 in combination with XENP16433 in anSEB-stimulated PBMC assay.

FIG. 110 depicts fold change in IL-2 secretion in MLR reactionsincubated with either XENP16432 or XmAb23104 (in comparison toincubation with isotype control anti-RSV mAb).

FIG. 111 depicts CD45+ cell counts on Day 8 in huPBMC-engrafted NSG micedosed with XENP16432 or XmAb23104, and show that XmAb23104 inducesgreater leukocyte expansion than αPD-1 (XENP16432) alone.

FIG. 112 depicts change in body weight over time in huPBMC-engrafted NSGmice dosed with XENP16432 or XmAb23104, and show that XmAb23104 enhancedGVHD in comparison to αPD-1 (XENP16432) alone.

FIG. 113 depicts CD45+ cell counts on Day 21 in pp65-MCF7 andhuPBMC-engrafted NSG mice dosed with XENP16432 or XmAb23104, and showthat XmAb23104 induces greater leukocyte expansion than αPD-1(XENP16432) alone.

FIG. 114 depicts tumor volume on Day 21 in pp65-MCF7 andhuPBMC-engrafted NSG mice dosed with XENP16432 or XmAb23104.

FIG. 115 depicts tumor volume on Day 38 in pp65-MCF7 andhuPBMC-engrafted NSG mice dosed with XENP16432 or XmAb23104, and showthat XmAb23104 enhances allogeneic anti-tumor response over αPD-1(XENP16432) alone. p-value was determined using an unpaired t test.

FIG. 116 depicts tumor volume over time (post-huPBMC engraftment) inpp65-MCF7 and huPBMC-engrafted NSG mice dosed with XENP16432 orXmAb23104.

FIG. 117 depicts the sequences of XENP20053, a bottle opener format withthe αCTLA-4 as the Fab side ([αCTLA-4]_H3L0.22) and the PD-1 as the scFv(1G6_L1.94_H1.279). The CDRs are underlined, the scFv linker is doubleunderlined (in the sequences, the scFv linker is a positively chargedscFv (GKPGS)4 linker (SEQ ID NO: 26209), although as will be appreciatedby those in the art, this linker can be replaced by other linkers,including uncharged or negatively charged linkers, some of which aredepicted in FIG. 8), and the slashes indicate the border(s) of thevariable domains. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CRD locations may beslightly different depending on the numbering used as is shown in Table1, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the VH and VL domains using othernumbering systems.

FIG. 118 depicts CD45+ cell counts in NSG mice on Day 14 afterengraftment with human PBMCs and treatment with the indicated testarticles.

FIGS. 119A to 119J depicts the release of A) IFNγ, B) IL-1ß, C) IL-2, D)IL-4, E) IL-8, F) IL-6, G) IL-10, H) IL-12p70, I) IL-13, and J) TNFαfrom human PBMCs treated with PBS, plate-bound XmAb23104, solubleXmAb23104, and plate-bound anti-CD3 antibody (OKT3).

FIG. 120 depicts the sequences for XENP29154, which is in-house producedTGN1412.

FIG. 121A to 121J depicts the release of A) IFNγ, B) IL-1ß, C) IL-2, D)IL-4, E) IL-8, F) IL-10, G) IL-12p70, H) TNF, I) IL-6, and J) IL-13 fromhuman PBMCs treated with air-dried XmAb23104, air-dried XENP15074(isotype control), air-dried XENP29154 (positive control), and air-driedanti-CD3 mAb (OKT3; positive control).

FIGS. 122A and 122B depicts sensorgrams showing binding of XmAb23104 toA) human PD-1 and B) cynomolgus PD-1.

FIGS. 123A and 123B depict sensorgrams showing binding of XmAb23104 toA) human ICOS and B) cynomolgus ICOS.

FIG. 124 depicts the equilibrium dissociation constants (KD),association rates (k_(a)), and dissociation rates (k_(d)) for binding ofXmAb23104 to human and cynomolgus PD-1 and ICOS.

FIG. 125 depicts sensorgrams from competition binding experiments ofPD-1 and ligands PD-L1 and PD-L2 with and without XmAb23104pre-incubation.

FIG. 126A to 126C depicts blocking of A) soluble PD-L1, B) solublePD-L2, and C) soluble ICOSL to cell surface-expressed PD-1 and ICOS onPD-1⁺ICOS⁺ HEK 293T cells.

FIG. 127 depicts sensorgrams showing binding of XmAb23104 (solid line)and human IgG comparator (an anti-CD19 antibody with a native IgG1constant region; dotted line) to A) human FcγRI, B) human FcγRIIb, C)human FcγRIIA (131H), D) human FcγRIIA (131R), E) human FcγRIIIA (158V),and F) human FcγRIIIA (158F).

FIG. 128 depicts sensorgrams showing binding of XmAb23104 (solid line)and human IgG comparator (an anti-CD19 antibody with a native IgG1constant region; dotted line) to A) cynomolgus FcγRI, B) cynomolgusFcγRIIA, C) cynomolgus FcγRIIb, and D) cynomolgus RcγRIIIA

FIG. 129 depicts sensorgrams showing binding of XmAb23104 (solid line)and human IgG comparator (an anti-CD19 antibody with a native IgG1constant region; dotted line) to A) murine FcγRI, B) murine FcγRIIb, C)murine FcγRIII, and D) murine FcγRIV.

FIG. 130 depicts equilibrium dissociation constants (K_(D)) for bindingof XmAb23104 and XENP20896 to human, cynomolgus, and mouse FcRn at pH6.0.

FIG. 131 depicts sensorgrams showing binding of XmAb23104 and XENP20896to human, cynomolgus, and mouse FcRn (1000, 500, 250, and 125 nM) at pH6.0.

FIG. 132 depicts in-tandem BLI experiment showing biosensors loaded withICOS and dipped into XmAb23104 or buffer followed by a final dip intoPD-1 antigen.

FIG. 133 depicts dose-dependent promotion of IL-2 secretion fromSEB-stimulated human PBMCs (from 19 different donors) by XmAb23104.

FIG. 134 depicts dose-dependent promotion of IFNγ secretion fromSEB-stimulated human PBMCs (from 19 different donors) by XmAb23104.

FIG. 135 depicts dose-dependent promotion of IFNγ secretion fromSEB-stimulated cynomolgus PBMCs (from 10 different donors) by XmAb23104.

FIG. 136 depicts tumor volume over time (post-huPBMC engraftment) inpp65-MCF7 and huPBMC-engrafted NSG mice dosed with XENP16432 orindicated concentrations of XmAb23104.

FIGS. 137A and 137B depict human CD45 cell counts on A) Day 14 and B)Day 21 in pp65-MCF7 and huPBMC-engrafted NSG mice dosed with XENP16432or indicated concentrations of XmAb23104. *denotes P<0.05, unpairedStudent t test, XmAb23104 or anti-PD-1 XENP16432-treated groups comparedto PBS-treated group. † denotes P<0.05, unpaired Student t test,XmAb23104-treated groups compared to XENP16432-treated group. Data werelog transformed prior to statistical analysis.

FIG. 138 depicts the sequences for JTX-2011, produced in-house asXENP23058.

FIG. 139 depicts the sequences for XENP23057, an anti-ICOS mAb based onJTX-2011 with E233P/L234V/L235A/G236del/S267K ablation variants.

FIGS. 140A to 140D depicts A) human CD45⁺ cell, B) human CD3⁺ T cell, C)human CD4⁺ T cell, and D) human CD8⁺ T cell counts in huPBMC-engraftedNSG mice on Day 21 after dosing with the indicated test articles.

DETAILED DESCRIPTION OF THE INVENTION I. Nomenclature

The bispecific antibodies of the invention are listed in severaldifferent formats. Each polypeptide is given a unique “XENP” number,although as will be appreciated in the art, a longer sequence mightcontain a shorter one. For example, the heavy chain of the scFv sidemonomer of a bottle opener format for a given sequence will have a firstXENP number, while the scFv domain will have a different XENP number.Some molecules have three polypeptides, so the XENP number, with thecomponents, is used as a name. Thus, the molecule XENP, which is inbottle opener format, comprises three sequences, generally referred toas “XENP23104-HC-Fab”, XENP23104 HC-scFv” and “XENP23104 LC” orequivalents, although one of skill in the art would be able to identifythese easily through sequence alignment. These XENP numbers are in thesequence listing as well as identifiers, and used in the Figures. Inaddition, one molecule, comprising the three components, gives rise tomultiple sequence identifiers. For example, the listing of the Fabmonomer has the full length sequence, the variable heavy sequence andthe three CDRs of the variable heavy sequence; the light chain has afull length sequence, a variable light sequence and the three CDRs ofthe variable light sequence; and the scFv-Fc domain has a full lengthsequence, an scFv sequence, a variable light sequence, 3 light CDRs, ascFv linker, a variable heavy sequence and 3 heavy CDRs; note that allmolecules herein with a scFv domain use a single charged scFv linker(+H), although others can be used. In addition, the naming nomenclatureof particular variable domains uses a “Hx.xx_Ly.yy” type of format, withthe numbers being unique identifiers to particular variable chainsequences. Thus, the variable domain of the scFv side of XENP23104(which binds PD-1) is “1G6_L1.194_H1.279”, which indicates that thevariable heavy domain H1.279 was combined with the light domain L1.194.In the case that these sequences are used as scFvs, the designation “1G6L1.194_H1.279”, indicates that the variable heavy domain H1.279 wascombined with the light domain L1.194 and is in vl-linker-vhorientation, from N- to C-terminus. This molecule with the identicalsequences of the heavy and light variable domains but in the reverseorder would be named “1G6_H1.279_L1.194”. Similarly, differentconstructs may “mix and match” the heavy and light chains as will beevident from the sequence listing and the Figures.

II. Incorporation of Materials

A. Figures and Legends

Specifically incorporated by reference are the Figures, Legends andSequences from U.S. Ser. No. 62/479,723 and the Figures, Legends andSequences from U.S. Ser. No. 15/623,314. In addition, the claims fromU.S. Ser. No. 62/479,723 are additionally specifically incorporated.

B. Sequences

Target Antigens: The sequence of human PD-1 (sp|Q15116) is SEQ ID NO:26226. The sequence of human PD-1, extracellular domain(sp|Q15116|21-170) is SEQ ID NO: 26227. The sequence of Macacafascicularis PD-1 (tr|B0LAJ3) is SEQ ID NO: 26228. The sequence ofMacaca fascicularis PD-1, extracellular domain (predicted)(tr|B0LAJ3|21-170) is SEQ ID NO: 26229. The sequence of human CTLA-4(sp|P16410) is SEQ ID NO: 26230. The sequence of human CTLA-4,extracellular domain (sp|P16410|36-161) is SEQ ID NO: 26231. Thesequence of Macaca fascicularis CTLA-4 (tr|G7PL88) is SEQ ID NO: 26232.The sequence of Macaca fascicularis CTLA-4, extracellular domain(predicted) (tr|G7PL88) is SEQ ID NO: 26233. The sequence of human LAG-3(sp|P18627) is SEQ ID NO: 26234. The sequence of human LAG-3,extracellular domain (sp|P18627|29-450) is SEQ ID NO: 26235. Thesequence of Macaca fascicularis LAG-3 (predicted)(gi|544467815|ref|XP_005570011.1) is SEQ ID NO: 26236. The sequence ofMacaca fascicularis LAG-3, extracellular domain (predicted)(gi|544467815|ref|XP_005570011.1129-450) is SEQ ID NO: 26237. Thesequence of human TIM-3 (sp|Q8TDQ0) is SEQ ID NO: 26238. The sequence ofhuman TIM-3, extracellular domain (sp|Q8TDQ0|22-202) is SEQ ID NO:26239. The sequence of Macaca fascicularis TIM-3 (predicted)(gi|355750365|gb|EHH54703.1) is SEQ ID NO: 26240. The sequence of Macacafascicularis TIM-3, extracellular domain (predicted)(gi|355750365|gb|EHH54703.1|22-203) is SEQ ID NO: 26241. The sequence ofhuman PD-L1 (sp|Q9NZQ7) is SEQ ID NO: 26242. The sequence of humanPD-L1, extracellular domain (sp|Q9NZQ7|19-238) is SEQ ID NO: 26243. Thesequence of Macaca fascicularis PD-L1 (predicted) (gb|XP_005581836.1) isSEQ ID NO: 26244. The sequence of Macaca fascicularis PD-L1,extracellular domain (predicted) (gb|XP_005581836.1|19-238) is SEQ IDNO: 26245. The sequence of human ICOS (sp|Q9Y6W8) is SEQ ID NO: 26246.The sequence of human ICOS, extracellular domain (sp|Q9Y6W8|21-140) isSEQ ID NO: 26247. The sequence of Macaca fascicularis ICOS(gi|544477053|ref|XP_005574075.1) is SEQ ID NO: 26248. The sequence ofMacaca fascicularis ICOS, extracellular domain (predicted)(gi|544477053|ref|XP_005574075.1121-140) is SEQ ID NO: 26249. Thesequence of human GITR (sp|Q9Y5U5) is SEQ ID NO: 26250. The sequence ofhuman GITR, extracellular domain (sp|Q9Y5U5|26-162) is SEQ ID NO: 26251.The sequence of Macaca fascicularis GITR (predicted)(ref|XP_005545180.1) is SEQ ID NO: 26252. The sequence of Macacafascicularis GITR, extracellular domain (predicted)(ref|XP_005545180.1|26-162) is SEQ ID NO: 26253. The sequence of humanOX40 (sp|P43489) is SEQ ID NO: 26254. The sequence of human OX40,extracellular domain (sp|P43489|29-214) is SEQ ID NO: 26255. Thesequence of Macaca fascicularis OX40 (predicted) (ref|XP_005545179.1) isSEQ ID NO: 26256. The sequence of Macaca fascicularis OX40,extracellular domain (predicted) (ref|XP_005545179.1|29-214) is SEQ IDNO: 26257. The sequence of human 4-1BB (sp|Q07011) is SEQ ID NO: 26258.The sequence of human 4-1BB, extracellular domain (sp|Q07011|24-186) isSEQ ID NO: 26259. The sequence of Macaca fascicularis 4-1BB (predicted)(ref|XP_005544945.1) is SEQ ID NO: 26260. The sequence of Macacafascicularis 4-1BB, extracellular domain (predicted)(ref|XP_005544945.1124-186) is SEQ ID NO: 26261.

ICOS binding domains: In addition the sequences shown in FIG. 19, FIG.20 and FIG. 24, SEQ ID NO:27869-28086 contain a number of ICOS Fabsequences (heavy chain VH1-CH1 and light chain VL1-CL) as indicated inthe naming nomenclature. Reference for the CDRs and for the junctionbetween the variable junctions is shown in FIG. 40 of U.S. Ser. No.62/479,723 (hereby incorporated by reference as well as the Legend),although from the SEQ listing one of skill in the art will be able toascertain the CDRs (see Table 1 for numbering and/or through sequencealignment) as well as for the junctions (e.g. heavy chain CH1 generallystarts with the sequence “ASTK . . . ” (SEQ ID NO: 30380) and lightchain constant domain generally starts with “RTVA . . . ” (SEQ ID NO:30381). SEQ ID NO:28087-28269 show the three sequences for “one armedmAb” (FIG. 2N; Fab-Fc, Fc only and light chain) as shown in the namingnomenclature. Reference for the CDRs and for the junction between thevariable junctions is shown in FIG. 41 of U.S. Ser. No. 62/479,723(hereby incorporated by reference as well as the Legend), although fromthe SEQ listing one of skill in the art will be able to ascertain theCDRs (see Table 1 for numbering and/or through sequence alignment) aswell as for the junctions (e.g. heavy chain CH1 generally starts withthe sequence “ASTK . . . ” (SEQ ID NO: 30380) and light chain constantdomain generally starts with “RTVA . . . ” (SEQ ID NO: 30381).Additional one armed ICOS molecules are shown in FIG. 68. SEQ IDNO:28549-28556 show some control antibodies (HC and LC) from which theFvs can be used as ICOS ABDs as well; reference for the CDRs and for thejunction between the variable junctions is shown in FIG. 44 of U.S. Ser.No. 62/479,723 (hereby incorporated by reference as well as the Legend),although from the SEQ listing one of skill in the art will be able toascertain the CDRs (see Table 1 for numbering and/or through sequencealignment) as well as for the junctions (e.g. heavy chain CH1 generallystarts with the sequence “ASTK . . . ” (SEQ ID NO: 30380) and lightchain constant domain generally starts with “RTVA . . . ” (SEQ ID NO:30381). SEQ ID NO:28557-28665 show some ICOS scFvs that find use incombination in the invention; reference for the CDRs and for thejunction between the variable junctions is shown in FIG. 45 of U.S. Ser.No. 62/479,723 (hereby incorporated by reference as well as the Legend),although from the SEQ listing one of skill in the art will be able toascertain the CDRs (see Table 1 for numbering and/or through sequencealignment) as well as for the junctions, as the scFvs utilize thecharged linker (GKPGS)4 between (SEQ ID NO: 26209) the vh and vldomains. Thus, suitable ICOS ABDs for use in combination with ABDs forcheckpoint receptors are shown in FIG. 19, FIG. 20, FIG. 24, FIG. 68 andFIG. 77 as well as SEQ ID NO: 27869-28086, 28087-28269, 27193-27335,28549-28556 and 28557-28665, and [ICOS]_H0.66_L0 and [ICOS]_H0_L0.

In addition, suitable ICOS X PD-1 bottle opener sequences include thosein FIG. 3 as well as those in SEQ ID NO:28270-28548, which correspond tothe sequences depicted in FIGS. 42 and 43 of U.S. Ser. No. 62/479,723(both of which are hereby incorporated by reference as well as theLegends and sequences therein), again using these figures to show CDRs,junctions, etc.

III. Overview

Therapeutic antibodies directed against immune immunomodulatoryinhibitors such as PD-1 are showing great promise in limitedcircumstances in the clinic for the treatment of cancer. Cancer can beconsidered as an inability of the patient to recognize and eliminatecancerous cells. In many instances, these transformed (e.g. cancerous)cells counteract immunosurveillance. There are natural controlmechanisms that limit T-cell activation in the body to preventunrestrained T-cell activity, which can be exploited by cancerous cellsto evade or suppress the immune response. Restoring the capacity ofimmune effector cells-especially T cells-to recognize and eliminatecancer is the goal of immunotherapy. The field of immuno-oncology,sometimes referred to as “immunotherapy” is rapidly evolving, withseveral recent approvals of T cell checkpoint inhibitory antibodies suchas Yervoy, Keytruda and Opdivo. These antibodies are generally referredto as “checkpoint inhibitors” because they block normally negativeregulators of T cell immunity. It is generally understood that a varietyof immunomodulatory signals, both costimulatory and coinhibitory, can beused to orchestrate an optimal antigen-specific immune response.

Checkpoint inhibitor monoclonal antibodies bind to immunomodulatoryinhibitor proteins such as PD-1, which under normal circumstancesprevent or suppress activation of cytotoxic T cells (CTLs). Byinhibiting the immunomodulatory protein, for example through the use ofantibodies that bind these proteins, an increased T cell responseagainst tumors can be achieved. That is, these cancer immunomodulatoryproteins suppress the immune response; when the proteins are blocked,for example using antibodies to the immunomodulatory protein, the immunesystem is activated, leading to immune stimulation, resulting intreatment of conditions such as cancer and infectious disease.Antibodies to either the PD-1 protein or its binding partner, PD-L1,leads to T cell activation.

Another area of current interest for harnessing the patient's immunesystem to fight disease involves the co-stimulation of T cells usingagonistic antibodies that bind to co-stimulatory proteins such as ICOS(Inducible T cell Co-Stimulator, also referred to as CD278) which adds apositive signal to overcome the negative signaling of the immunecheckpoint proteins on the T-cells. ICOS is a type I transmembraneprotein comprising an extracellular (Ig) V-like domain, and serves asthe receptor for the B7h co-stimulatory molecule.

Recent work shows that some tumor infiltrating lymphocytes (TILs)co-express PD-1 and ICOS (see Gros, J. Clinical Invest. 124(5):2246(2014)).

Bispecific antibodies, which can bind two different targetssimultaneously, offer the potential to improve the selectivity oftargeting TILs vs peripheral T cells, while also reducing cost oftherapy. The bivalent interaction of an antibody with two targets on acell surface should—in some cases—lead to a higher binding avidityrelative to a monovalent interaction with one target at a time. Becauseof this, normal bivalent antibodies tend to have high avidity for theirtarget on a cell surface. With bispecific antibodies, the potentialexists to create higher selectivity for cells that simultaneouslyexpress two different targets, utilizing the higher avidity afforded bysimultaneous binding to both targets.

Accordingly, the present invention provides bispecific immunomodulatoryantibodies, that bind to cells expressing the two antigens and methodsof activating T cells and/or NK cells to treat diseases such as cancerand infectious diseases, and other conditions where increased immuneactivity results in treatment.

Thus, the invention is directed, in some instances, to solving the issueof toxicity and expense of administering multiple antibodies byproviding bispecific antibodies that bind to two differentimmunomodulatory molecules (one a checkpoint receptor and the other acostimulatory receptor) on a single cell and advantageously requiringadministration of only one therapeutic substance.

Bispecific antibodies offer the opportunity to combine immuneimmunomodulatory blockade with costimulation in one molecule. However,it is not obvious what combination of immune immunomodulatory pluscostimulatory protein or what binding stoichiometry(monovalent+monovalent, monovalent+bivalent, etc.) would be efficacious.Here we identify bispecific antibodies that binding monovalently to acostimulatory protein (such as ICOS) and monovalent binding to acheckpoint receptor (such as PD-1) that are capable of inducing robust Tcell activation.

Surprisingly, while conventional wisdom states that monovalentantibodies do not result in agonism, the present work shows theunexpected results of agonism of the ICOS receptor with the monovalentbispecific antibodies of the invention. See Merchant et al., PNAS Jul.23 2013 E2987-E2996, “[w]hile initial screening of bivalent antibodiesproduced agonists of MET, engineering them into monovalent antibodiesproduces antagonists instead.” This positive result with only monovalentbinding to ICOS is unexpected because it is thought that at leastbivalent binding to a costimulatory protein is necessary to provide therequired level of receptor clustering on the cell surface for triggeringsignaling. As shown by Fos et al., J. Immunol. 2008:1969-1977, ICOSligation induces AKT phosphorylation. The studies here in use AKTphosphorylation as an indicator of ICOS agonism, and this effect is seenfor both “one armed ICOS” (see Example 5A(a)) and for bispecificantibodies that bind ICOS monovalently. As shown in Example 7 and inFIG. 70, the one-arm XENP20266 that only binds ICOS monovalentlypromotes more AKT phosphorylation than XENP16435, which binds ICOSbivalently (e.g. as a traditional mAb).

Accordingly, the present invention is directed to novel constructs toprovide heterodimeric, bispecific antibodies that allow binding to acheckpoint receptor as well as human ICOS.

Note that generally these bispecific antibodies are named“anti-PD-1×anti-ICOS”, or generally simplistically or for ease (and thusinterchangeably) as “PD-1×ICOS”, etc. for each pair.

The heterodimeric bispecific immunomodulatory antibodies of theinvention are useful to treat a variety of types of cancers. As will beappreciated by those in the art, in contrast to traditional monoclonalantibodies that bind to tumor antigens, or to the newer classes ofbispecific antibodies that bind, for example, CD3 and tumor antigens(such as described in U.S. Ser. No. 15/141,350, for example),immunomodulatory antibodies are used to increase the immune response butare not generally tumor specific in their action. That is, thebispecific immunomodulatory antibodies of the invention inhibit thesuppression of the immune system, generally leading to T cellactivation, which in turn leads to greater immune response to cancerouscells and thus treatment.

As discussed below, there are a variety of ways that T cell activationcan be measured. Functional effects of the bispecific immunomodulatoryantibodies on NK and T-cells can be assessed in vitro (and in some casesin vivo, as described more fully below) by measuring changes in thefollowing parameters: proliferation, cytokine release and cell-surfacemakers. For NK cells, increases in cell proliferation, cytotoxicity(ability to kill target cells as measured by increases in CD107a,granzyme, and perforin expression, or by directly measuring target cellskilling), cytokine production (e.g. IFN-γ and TNF), and cell surfacereceptor expression (e.g. CD25) is indicative of immune modulation, e.g.enhanced killing of cancer cells. For T-cells, increases inproliferation, increases in expression of cell surface markers ofactivation (e.g. CD25, CD69, CD137, and PD1), cytotoxicity (ability tokill target cells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFN,TNF-a, IL-10, IL-17A) are indicative of immune modulation, e.g. enhancedkilling of cancer cells. Accordingly, assessment of treatment can bedone using assays that evaluate one or more of the following: (i)increases in immune response, (ii) increases in activation of αβ and/orγ6 T cells, (iii) increases in cytotoxic T cell activity, (iv) increasesin NK and/or NKT cell activity, (v) alleviation of αβ and/or γδ T-cellsuppression, (vi) increases in pro-inflammatory cytokine secretion,(vii) increases in IL-2 secretion; (viii) increases in interferon-γproduction, (ix) increases in Th1 response, (x) decreases in Th2response, (xi) decreases or eliminates cell number and/or activity of atleast one of regulatory T cells, (xii) increases in IL-2 secretion.

Thus, in some embodiments the invention provides the use of bispecificimmunomodulatory antibodies to perform one or more of the following in asubject in need thereof: (a) upregulating pro-inflammatory cytokines;(b) increasing T-cell proliferation and/or expansion; (c) increasinginterferon-γ or TNF-α production by T-cells; (d) increasing IL-2secretion; (e) stimulating antibody responses; (f) inhibiting cancercell growth; (g) promoting antigenic specific T cell immunity; (h)promoting CD4+ and/or CD8+ T cell activation; (i) alleviating T-cellsuppression; (j) promoting NK cell activity; (k) promoting apoptosis orlysis of cancer cells; and/or (1) cytotoxic or cytostatic effect oncancer cells.

Accordingly, the present invention provides bispecific immunomodulatoryantibodies. There are a number of formats that can be used in thepresent invention, as generally shown in FIG. 2, many of which areheterodimeric (although not all, as DVD-Ig, for example).

The heterodimeric antibodies constructs are based on the self-assemblingnature of the two Fc domains of the heavy chains of antibodies, e.g. two“monomers” that assemble into a “dimer”. Heterodimeric antibodies aremade by altering the amino acid sequence of each monomer as more fullydiscussed below. Thus, the present invention is generally directed tothe creation of heterodimeric antibodies, which can co-engage twoantigens in several ways, relying on amino acid variants in the constantregions that are different on each chain to promote heterodimericformation and/or allow for ease of purification of heterodimers over thehomodimers.

Thus, the present invention provides bispecific immunomodulatoryantibodies. An ongoing problem in antibody technologies is the desirefor “bispecific” antibodies that bind to two (or more) differentantigens simultaneously, in general thus allowing the different antigensto be brought into proximity and resulting in new functionalities andnew therapies. In general, these antibodies are made by including genesfor each heavy and light chain into the host cells (generally, in thepresent invention, genes for two heavy chain monomers and a light chainas outlined herein). This generally results in the formation of thedesired heterodimer (A-B), as well as the two homodimers (A-A and B-B).However, a major obstacle in the formation of bispecific antibodies isthe difficulty in purifying the heterodimeric antibodies away from thehomodimeric antibodies and/or biasing the formation of the heterodimerover the formation of the homodimers.

To solve this issue, there are a number of mechanisms that can be usedto generate the heterodimers of the present invention. In addition, aswill be appreciated by those in the art, these mechanisms can becombined to ensure high heterodimerization. Thus, amino acid variantsthat lead to the production of heterodimeric antibodies are referred toas “heterodimerization variants”. As discussed below, heterodimerizationvariants can include steric variants (e.g. the “knobs and holes” or“skew” variants described below and the “charge pairs” variantsdescribed below) as well as “pI variants”, which allows purification ofhomodimers away from heterodimers.

One mechanism is generally referred to in the art as “knobs and holes”(“KIH”) or sometimes herein as “skew” variants, referring to amino acidengineering that creates steric influences to favor heterodimericformation and disfavor homodimeric formation can also optionally beused; this is sometimes referred to as “knobs and holes”; as describedin Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al.,J. Mol. Biol. 1997 270:26; U.S. Pat. No. 8,216,805, US 2012/0149876, allof which are hereby incorporated by reference in their entirety. TheFigures identify a number of “monomer A—monomer B” pairs that include“knobs and holes” amino acid substitutions. In addition, as described inMerchant et al., Nature Biotech. 16:677 (1998), these “knobs and hole”mutations can be combined with disulfide bonds to skew formation toheterodimerization. Of use in the present invention areT366S/L368A/Y407V paired with T366W, as well as this variant with abridging disulfide, T366S/L368A/Y407V/Y349C paired with T366W/S354C,particularly in combination with other heterodimerization variantsincluding pI variants as outlined below.

An additional mechanism that finds use in the generation ofheterodimeric antibodies is sometimes referred to as “electrostaticsteering” or “charge pairs” as described in Gunasekaran et al., J. Biol.Chem. 285(25):19637 (2010), hereby incorporated by reference in itsentirety. This is sometimes referred to herein as “charge pairs”. Inthis embodiment, electrostatics are used to skew the formation towardsheterodimerization. As those in the art will appreciate, these may alsohave an effect on pI, and thus on purification, and thus could in somecases also be considered pI variants. However, as these were generatedto force heterodimerization and were not used as purification tools,they are classified as “steric variants”. These include, but are notlimited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g. theseare “monomer corresponding sets) and C220E/P228E/368E paired withC220R/E224R/P228R/K409R and others shown in the Figures.

In the present invention, in some embodiments, pI variants are used toalter the pI of one or both of the monomers and thus allowing theisoelectric purification of A-A, A-B and B-B dimeric proteins.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric proteins; one relies on the useof pI variants, such that each monomer has a different pI, thus allowingthe isoelectric purification of A-A, A-B and B-B dimeric proteins.Alternatively, some scaffold formats, such as the “triple F” or “bottleopener” format, also allows separation on the basis of size. As isfurther outlined below, it is also possible to “skew” the formation ofheterodimers over homodimers. Thus, a combination of stericheterodimerization variants and pI or charge pair variants findparticular use in the invention. Additionally, as more fully outlinedbelow, scaffolds that utilize scFv(s) such as the Triple F format caninclude charged scFv linkers (either positive or negative), that give afurther pI boost for purification purposes. As will be appreciated bythose in the art, some Triple F formats are useful with just chargedscFv linkers and no additional pI adjustments, although the inventiondoes provide the use of skew variants with charged scFv linkers as well(and combinations of Fc, FcRn and KO variants discussed herein).

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As is outlined more fully below, the pI changesof either or both monomers can be done by removing or adding a chargedresidue (e.g. a neutral amino acid is replaced by a positively ornegatively charged amino acid residue, e.g. glycine to glutamic acid),changing a charged residue from positive or negative to the oppositecharge (aspartic acid to lysine) or changing a charged residue to aneutral residue (e.g. loss of a charge; lysine to serine). A number ofthese variants are shown in the Figures. In addition, suitable pIvariants for use in the creation of heterodimeric antibodies herein arethose that are isotypic, e.g. importing pI variants from different IgGisotypes such that pI is changed without introducing significantimmunogenicity; see FIG. 29 from US Publication No. 20140288275, herebyincorporated by reference in its entirety.

Accordingly, in this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseits pI (wt A− +B or wt A− −B), or by increasing one region anddecreasing the other region (A+ −B− or A− B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions of antibodiesthat are directed to altering the isoelectric point (pI) of at leastone, if not both, of the monomers of a dimeric protein to form “pIheterodimers” (when the protein is an antibody, these are referred to as“pI antibodies”) by incorporating amino acid substitutions (“pIvariants” or “pI substitutions”) into one or both of the monomers. Asshown herein, the separation of the heterodimers from the two homodimerscan be accomplished if the pIs of the two monomers differ by as littleas 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use inthe present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the scFv and Fab of interest. Thatis, to determine which monomer to engineer or in which “direction” (e.g.more positive or more negative), the Fv sequences of the two targetantigens are calculated and a decision is made from there. As is knownin the art, different Fvs will have different starting pIs which areexploited in the present invention. In general, as outlined herein, thepIs are engineered to result in a total pI difference of each monomer ofat least about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein. Furthermore, as will be appreciated by those in the art andoutlined herein, in some cases (depending on the format) heterodimerscan be separated from homodimers on the basis of size (e.g. Molecularweight). For example, as shown in some embodiments of FIG. 2, someformats result in homodimers and heterodimers with different sizes (e.g.for bottle openers, one homodimer is a “dual scFv” format, one homodimeris a standard antibody, and the heterodimer has one Fab and one scFv).

In the case where pI variants are used to achieve purified heterodimersover homodimers, by using the constant region(s) of the heavy chain(s),a more modular approach to designing and purifying multispecificproteins, including antibodies, is provided. Thus, in some embodiments,heterodimerization variants (including skew and purificationheterodimerization variants) are not included in the variable regions,such that each individual antibody must be engineered. In addition, insome embodiments, the possibility of immunogenicity resulting from thepI variants is significantly reduced by importing pI variants fromdifferent IgG isotypes such that pI is changed without introducingsignificant immunogenicity. Thus, an additional problem to be solved isthe elucidation of low pI constant domains with high human sequencecontent, e.g. the minimization or avoidance of non-human residues at anyparticular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of bispecific antibodies, as the ability toeither eliminate, minimize and distinguish when homodimers are presentis significant. Similarly, the ability to reliably test thereproducibility of the heterodimeric protein production is important.

First and second antigens of the invention are herein referred to asantigen-1 and antigen-2 respectively, with one being a costimulatoryreceptor and one being a checkpoint receptor. One heterodimeric scaffoldthat finds particular use in the present invention is the “triple f” or“bottle opener” scaffold format. In this embodiment, one heavy chain ofthe antibody contains an single chain fv (“scfv”, as defined below) andthe other heavy chain is a “regular” fab format, comprising a variableheavy chain and a light chain. This structure is sometimes referred toherein as “triple f” format (scfv-fab-fc) or the “bottle-opener” format,due to a rough visual similarity to a bottle-opener (see FIG. 2). Thetwo chains are brought together by the use of amino acid variants in theconstant regions (e.g. the Fc domain and/or the hinge region) thatpromote the formation of heterodimeric antibodies as is described morefully below.

There are several distinct advantages to the present “triple F” format.As is known in the art, antibody analogs relying on two scFv constructsoften have stability and aggregation problems, which can be alleviatedin the present invention by the addition of a “regular” heavy and lightchain pairing. In addition, as opposed to formats that rely on two heavychains and two light chains, there is no issue with the incorrectpairing of heavy and light chains (e.g. heavy 1 pairing with light 2,etc.)

Furthermore, as outlined herein, additional amino acid variants may beintroduced into the bispecific antibodies of the invention, to addadditional functionalities. For example, amino acid changes within theFc region can be added (either to one monomer or both) to facilitateincreased ADCC or CDC (e.g. altered binding to Fcγ receptors) as well asto increase binding to FcRn and/or increase serum half-life of theresulting molecules. As is further described herein and as will beappreciated by those in the art, any and all of the variants outlinedherein can be optionally and independently combined with other variants.

Similarly, another category of functional variants are “Fcγ ablationvariants” or “Fc knock out (FcKO or KO) variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, it is generallydesirable to ablate FcγRIIIa binding to eliminate or significantlyreduce ADCC activity. Suitable ablation variants are shown in FIG. 6.

IV. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding are those shown in FIG. 6.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity. As is discussed herein, many embodiments of theinvention ablate ADCC activity entirely.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen as discussedherein. Thus, a “immunomodulatoryantigen binding domain” binds a targetimmunomodulatoryantigen as outlined herein. As is known in the art,these CDRs are generally present as a first set of variable heavy CDRs(vhCDRs or VHCDRs) and a second set of variable light CDRs (vlCDRs orV_(L)CDRs), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 for theheavy chain and vlCDR1, vlCDR2 and vlCDR3 for the light. The CDRs arepresent in the variable heavy and variable light domains, respectively,and together form an Fv region. Thus, in some cases, the six CDRs of theantigen binding domain are contributed by a variable heavy and variablelight chain. In a “Fab” format, the set of 6 CDRs are contributed by twodifferent polypeptide sequences, the variable heavy domain (vh or VH;containing the vhCDR1, vhCDR2 and vhCDR3) and the variable light domain(vl or VL; containing the vlCDR1, vlCDR2 and vlCDR3), with theC-terminus of the vh domain being attached to the N-terminus of the CH1domain of the heavy chain and the C-terminus of the vl domain beingattached to the N-terminus of the constant light domain (and thusforming the light chain). In a scFv format, the vh and vl domains arecovalently attached, generally through the use of a linker as outlinedherein, into a single polypeptide sequence, which can be either(starting from the N-terminus) vh-linker-vl or vl-linker-vh, with theformer being generally preferred (including optional domain linkers oneach side, depending on the format used.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233- or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”, for example theIgG1/2 hybrid can be included. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Variant protein can referto the variant protein itself, compositions comprising the proteinvariant, or the DNA sequence that encodes it.

Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification, and “immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,N434S or 434S is an Fc variant with the substitution serine at position434 relative to the parent Fc polypeptide, wherein the numbering isaccording to the EU index. Likewise, M428L/N434S defines an Fc variantwith the substitutions M428L and N434S relative to the parent Fcpolypeptide. The identity of the WT amino acid may be unspecified, inwhich case the aforementioned variant is referred to as 428L/434S. It isnoted that the order in which substitutions are provided is arbitrary,that is to say that, for example, 428L/434S is the same Fc variant asM428L/N434S, and so on. For all positions discussed in the presentinvention that relate to antibodies, unless otherwise noted, amino acidposition numbering is according to the EU index. The EU index or EUindex as in Kabat or EU numbering scheme refers to the numbering of theEU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,hereby entirely incorporated by reference.) The modification can be anaddition, deletion, or substitution. Substitutions can include naturallyoccurring amino acids and, in some cases, synthetic amino acids.Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO 03/073238;US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, etal., (2002), PICAS United States of America 99:11020-11024; and, L.Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated byreference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single ABD. Aswill be appreciated by those in the art, these generally are made up oftwo chains, or can be combined (generally with a linker as discussedherein) to form an scFv. In some cases, for example in the “central-Fv”and “DVD-Ig” formats, an “extra” vh and vl domain is added that servesas a scFv but where the vh and vl domains are not linked using a scFvlinker between them.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N-to C-terminus (vh-linker-vl orvl-linker-vh).

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life, are shown in the Figure Legend ofFIG. 83.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain and in some cases, part ofthe hinge. Thus Fc refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, the last three constant regionimmunoglobulin domains of IgE and IgM, and the flexible hinge N-terminalto these domains. For IgA and IgM, Fc may include the J chain. For IgG,the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3)and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR receptors or to the FcRn receptor.

By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portionof an antibody.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as a bindingmoiety to a target protein, as described herein. In some cases, onemonomer of the heterodimeric antibody comprises an antibody heavy chain(either including an scFv or further including a light chain) and theother monomer is a Fc fusion, comprising a variant Fc domain and aligand. In some embodiments, these “half antibody-half fusion proteins”are referred to as “Fusionbodies”.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. Suitable target antigens are described below.

By “strandedness” in the context of the monomers of the heterodimericantibodies of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities. Similarly,for “skew” variants that come in pairs of a set as more fully outlinedbelow, the skilled artisan will consider pI in deciding into whichstrand or monomer that incorporates one set of the pair will go, suchthat pI separation is maximized using the pI of the skews as well.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V.kappa., V.lamda., and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. “Recombinant” means the antibodiesare generated using recombinant nucleic acid techniques in exogeneoushost cells.

“Percent (%) amino acid sequence identity” with respect to a proteinsequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific (parental) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.One particular program is the ALIGN-2 program outlined at paragraphs[0279] to [0280] of US Pub. No. 20160244525, hereby incorporated byreference.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the parental amino acid sequence iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence,” or thelength of the parental sequence, whichever is the shortest. The resultis expressed in percent identity.

In some embodiments, two or more amino acid sequences are at least 50%,60%, 70%, 80%, or 90% identical. In some embodiments, two or more aminoacid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using a Biacoreassay.

V. Antibodies

The present invention relates to the generation of bispecificimmunomodulatory antibodies that bind two different immunomodulatoryantigens as discussed herein. As is discussed below, the term “antibody”is used generally. Antibodies that find use in the present invention cantake on a number of formats as described herein.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to bispecific antibodies that generally are based on the IgGclass, which has several subclasses, including, but not limited to IgG1,IgG2, IgG3, and IgG4. In general, IgG1, IgG2 and IgG4 are used morefrequently than IgG3. It should be noted that IgG1 has differentallotypes with polymorphisms at 356 (D or E) and 358 (L or M). Thesequences depicted herein use the 356D/358M allotype, however the otherallotype is included herein. That is, any sequence inclusive of an IgG1Fc domain included herein can have 356E/358L replacing the 356D/358Mallotype.

In addition, many of the sequences herein have at least one of thecysteines at position 220 replaced by a serine; generally, this is theon the “scFv monomer” side for most of the sequences depicted herein,although it can also be on the “Fab monomer” side, or both, to reducedisulfide formation. Specifically included within the sequences hereinare one or both of these cysteines replaced (C220S).

Thus, “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. It should be understood that therapeuticantibodies can also comprise hybrids of isotypes and/or subclasses. Forexample, as shown in US Publication 2009/0163699, incorporated byreference, the present invention covers pI engineering of IgG1/G2hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

A useful comparison of CDR numbering is as below, see Lafranc et al.,Dev. Comp. Immunol. 27(1):55-77 (2003):

TABLE 1

Kabat+ IMGT Kabat AbM Chothia Contact Xencor

Chothia

vhCDR1 26-35 27-38 31-35 26-35 26-32 30-35 27-35vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58 54-61vhCDR3 95-102 105-117 95-102 95-102 95-102 93-101 103-116vlCDR1 24-34 27-38 24-34 24-34 24-34 30-36 27-38vlCDR2 50-56 56-65 50-56 50-56 50-56 46-55 56-62vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96 97-105

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g., Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used (for example when Fabs areused), or on a single polypeptide chain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or C_(κ).

Another region of interest for additional substitutions, outlined below,is the Fc region.

Thus, the present invention provides different antibody domains. Asdescribed herein and known in the art, the heterodimeric antibodies ofthe invention comprise different domains within the heavy and lightchains, which can be overlapping as well. These domains include, but arenot limited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3domain, the hinge domain, the heavy constant domain (CH1-hinge-Fc domainor CH1-hinge-CH2-CH3), the variable heavy domain, the variable lightdomain, the light constant domain, Fab domains and scFv domains.

Thus, the “Fc domain” includes the —CH2-CH3 domain, and optionally ahinge domain. In the embodiments herein, when a scFv is attached to anFc domain, it is the C-terminus of the scFv construct that is attachedto all or part of the hinge of the Fc domain; for example, it isgenerally attached to the sequence EPKS which is the beginning of thehinge. The heavy chain comprises a variable heavy domain and a constantdomain, which includes a CH1-optional hinge-Fc domain comprising aCH2-CH3. The light chain comprises a variable light chain and the lightconstant domain. A scFv comprises a variable heavy chain, an scFvlinker, and a variable light domain. In most of the constructs andsequences outlined herein, C-terminus of the variable light chain isattached to the N-terminus of the scFv linker, the C-terminus of whichis attached to the N-terminus of a variable heavy chain(N-vh-linker-vl-C) although that can be switched (N-vl-linker-vh-C).Some embodiments of the invention comprise at least one scFv domain,which, while not naturally occurring, generally includes a variableheavy domain and a variable light domain, linked together by a scFvlinker. As outlined herein, while the scFv domain is generally from N-to C-terminus oriented as vh-scFv linker-vl, this can be reversed forany of the scFv domains (or those constructed using vh and vl sequencesfrom Fabs), to vl-scFv linker-vh, with optional linkers at one or bothends depending on the format (see generally FIG. 2).

As shown herein, there are a number of suitable scFv linkers that can beused, including traditional peptide bonds, generated by recombinanttechniques. The linker peptide may predominantly include the followingamino acid residues: Gly, Ser, Ala, or Thr. The linker peptide shouldhave a length that is adequate to link two molecules in such a way thatthey assume the correct conformation relative to one another so thatthey retain the desired activity. In one embodiment, the linker is fromabout 1 to 50 amino acids in length, preferably about 1 to 30 aminoacids in length. In one embodiment, linkers of 1 to 20 amino acids inlength may be used, with from about 5 to about 10 amino acids findinguse in some embodiments. Useful linkers include glycine-serine polymers,including for example (GS)n, (GSGGS)n (SEQ ID NO: 30382), (GGGGS)n (SEQID NO: 30383), and (GGGS)n (SEQ ID NO: 30384, where n is an integer ofat least one (and generally from 3 to 4), glycine-alanine polymers,alanine-serine polymers, and other flexible linkers. Alternatively, avariety of nonproteinaceous polymers, including but not limited topolyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol, may find useas linkers, that is may find use as linkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. While any suitable linker canbe used, many embodiments utilize a glycine-serine polymer, includingfor example (GS)n, (GSGGS)n (SEQ ID NO: 30382), (GGGGS)n (SEQ ID NO:30383), and (GGGS)n (SEQ ID NO: 30384), where n is an integer of atleast one (and generally from 3 to 4 to 5) as well as any peptidesequence that allows for recombinant attachment of the two domains withsufficient length and flexibility to allow each domain to retain itsbiological function. In some cases, and with attention being paid to“strandedness”, as outlined below, charged domain linkers, as used insome embodiments of scFv linkers can be used.

In some embodiments, the scFv linker is a charged scFv linker, a numberof which are shown in FIG. 8. Accordingly, the present invention furtherprovides charged scFv linkers, to facilitate the separation in pIbetween a first and a second monomer. That is, by incorporating acharged scFv linker, either positive or negative (or both, in the caseof scaffolds that use scFvs on different monomers), this allows themonomer comprising the charged linker to alter the pI without makingfurther changes in the Fc domains. These charged linkers can besubstituted into any scFv containing standard linkers. Again, as will beappreciated by those in the art, charged scFv linkers are used on thecorrect “strand” or monomer, according to the desired changes in pI. Forexample, as discussed herein, to make triple F format heterodimericantibody, the original pI of the Fv region for each of the desiredantigen binding domains are calculated, and one is chosen to make anscFv, and depending on the pI, either positive or negative linkers arechosen.

Charged domain linkers can also be used to increase the pI separation ofthe monomers of the invention as well, and thus those included in FIG. 8can be used in any embodiment herein where a linker is utilized.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be engineered to produceheterodimers, such as pI engineering. Other antibody fragments that canbe used include fragments that contain one or more of the CH1, CH2, CH3,hinge and CL domains of the invention that have been pI engineered. Inparticular, the formats depicted in FIG. 1 are antibodies, usuallyreferred to as “heterodimeric antibodies”, meaning that the protein hasat least two associated Fc sequences self-assembled into a heterodimericFc domain and at least two Fv regions, whether as Fabs or as scFvs.

A. Chimeric and Humanized Antibodies

In some embodiments, the antibodies herein can be derived from a mixturefrom different species, e.g. a chimeric antibody and/or a humanizedantibody. In general, both “chimeric antibodies” and “humanizedantibodies” refer to antibodies that combine regions from more than onespecies. For example, “chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human. “Humanized antibodies” generally refer tonon-human antibodies that have had the variable-domain framework regionsswapped for sequences found in human antibodies. Generally, in ahumanized antibody, the entire antibody, except the CDRs, is encoded bya polynucleotide of human origin or is identical to such an antibodyexcept within its CDRs. The CDRs, some or all of which are encoded bynucleic acids originating in a non-human organism, are grafted into thebeta-sheet framework of a human antibody variable region to create anantibody, the specificity of which is determined by the engrafted CDRs.The creation of such antibodies is described in, e.g., WO 92/11018,Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science239:1534-1536, all entirely incorporated by reference. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213, all entirelyincorporated by reference). The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region. Humanized antibodies can also be generatedusing mice with a genetically engineered immune system. Roque et al.,2004, Biotechnol. Prog. 20:639-654, entirely incorporated by reference.A variety of techniques and methods for humanizing and reshapingnon-human antibodies are well known in the art (See Tsurushita &Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biologyof B Cells, 533-545, Elsevier Science (USA), and references citedtherein, all entirely incorporated by reference). Humanization methodsinclude but are not limited to methods described in Jones et al., 1986,Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329;Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, ProcNatl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160:1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Prestaet al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc.Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng11:321-8, all entirely incorporated by reference. Humanization or othermethods of reducing the immunogenicity of nonhuman antibody variableregions may include resurfacing methods, as described for example inRoguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, entirelyincorporated by reference.

In certain embodiments, the antibodies of the invention comprise a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene. For example, suchantibodies may comprise or consist of a human antibody comprising heavyor light chain variable regions that are “the product of” or “derivedfrom” a particular germline sequence. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally-occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a humanized antibodytypically is at least 90% identical in amino acids sequence to an aminoacid sequence encoded by a human germline immunoglobulin gene andcontains amino acid residues that identify the antibody as being derivedfrom human sequences when compared to the germline immunoglobulin aminoacid sequences of other species (e.g., murine germline sequences). Incertain cases, a humanized antibody may be at least 95, 96, 97, 98 or99%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a humanized antibody derived from aparticular human germline sequence will display no more than 10-20 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene (prior to the introduction of any skew, pIand ablation variants herein; that is, the number of variants isgenerally low, prior to the introduction of the variants of theinvention). In certain cases, the humanized antibody may display no morethan 5, or even no more than 4, 3, 2, or 1 amino acid difference fromthe amino acid sequence encoded by the germline immunoglobulin gene(again, prior to the introduction of any skew, pI and ablation variantsherein; that is, the number of variants is generally low, prior to theintroduction of the variants of the invention).

In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

VI. Heterodimeric Antibodies

Accordingly, in some embodiments the present invention providesheterodimeric immunomodulatory antibodies that rely on the use of twodifferent heavy chain variant Fc sequences, that will self-assemble toform heterodimeric Fc domains and heterodimeric antibodies.

The present invention is directed to novel constructs to provideheterodimeric antibodies that allow binding to more than oneimmunomodulatory antigen or ligand, e.g. to allow for bispecificbinding. The heterodimeric antibody constructs are based on theself-assembling nature of the two Fc domains of the heavy chains ofantibodies, e.g. two “monomers” that assemble into a “dimer”.Heterodimeric antibodies are made by altering the amino acid sequence ofeach monomer as more fully discussed below. Thus, the present inventionis generally directed to the creation of heterodimeric immunomodulatoryantibodies which can co-engage antigens in several ways, relying onamino acid variants in the constant regions that are different on eachchain to promote heterodimeric formation and/or allow for ease ofpurification of heterodimers over the homodimers.

Thus, the present invention provides bispecific antibodies. An ongoingproblem in antibody technologies is the desire for “bispecific”antibodies that bind to two different antigens simultaneously, ingeneral thus allowing the different antigens to be brought intoproximity and resulting in new functionalities and new therapies. Ingeneral, these antibodies are made by including genes for each heavy andlight chain into the host cells. This generally results in the formationof the desired heterodimer (A-B), as well as the two homodimers (A-A andB-B (not including the light chain heterodimeric issues)). However, amajor obstacle in the formation of bispecific antibodies is thedifficulty in purifying the heterodimeric antibodies away from thehomodimeric antibodies and/or biasing the formation of the heterodimerover the formation of the homodimers.

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art, these mechanisms can be combined toensure high heterodimerization. Thus, amino acid variants that lead tothe production of heterodimers are referred to as “heterodimerizationvariants”. As discussed below, heterodimerization variants can includesteric variants (e.g. the “knobs and holes” or “skew” variants describedbelow and the “charge pairs” variants described below) as well as “pIvariants”, which allows purification of homodimers away fromheterodimers. As is generally described in WO2014/145806, herebyincorporated by reference in its entirety and specifically as below forthe discussion of “heterodimerization variants”, useful mechanisms forheterodimerization include “knobs and holes” (“KIH”; sometimes herein as“skew” variants (see discussion in WO2014/145806), “electrostaticsteering” or “charge pairs” as described in WO2014/145806, pI variantsas described in WO2014/145806, and general additional Fc variants asoutlined in WO2014/145806 and below.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric antibodies; one relies on theuse of pI variants, such that each monomer has a different p1, thusallowing the isoelectric purification of A-A, A-B and B-B dimericproteins. Alternatively, some scaffold formats, such as the “triple F”format, also allows separation on the basis of size. As is furtheroutlined below, it is also possible to “skew” the formation ofheterodimers over homodimers. Thus, a combination of stericheterodimerization variants and pI or charge pair variants findparticular use in the invention.

In general, embodiments of particular use in the present invention relyon sets of variants that include skew variants, that encourageheterodimerization formation over homodimerization formation, coupledwith pI variants, which increase the pI difference between the twomonomers.

Additionally, as more fully outlined below, depending on the format ofthe heterodimer antibody, pI variants can be either contained within theconstant and/or Fc domains of a monomer, or charged linkers, eitherdomain linkers or scFv linkers, can be used. That is, scaffolds thatutilize scFv(s) such as the Triple F format can include charged scFvlinkers (either positive or negative), that give a further pI boost forpurification purposes. As will be appreciated by those in the art, someTriple F formats are useful with just charged scFv linkers and noadditional pI adjustments, although the invention does provide pIvariants that are on one or both of the monomers, and/or charged domainlinkers as well. In addition, additional amino acid engineering foralternative functionalities may also confer pI changes, such as Fc, FcRnand KO variants.

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As discussed, the pI changes of either or bothmonomers can be done by removing or adding a charged residue (e.g. aneutral amino acid is replaced by a positively or negatively chargedamino acid residue, e.g. glycine to glutamic acid), changing a chargedresidue from positive or negative to the opposite charge (e.g. asparticacid to lysine) or changing a charged residue to a neutral residue (e.g.loss of a charge; lysine to serine.). A number of these variants areshown in the Figures.

Accordingly, this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseits pI (wt A− +B or wt A− −B), or by increasing one region anddecreasing the other region (A+ −B− or A− B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions of antibodiesthat are directed to altering the isoelectric point (pI) of at leastone, if not both, of the monomers of a dimeric protein to form “pIantibodies” by incorporating amino acid substitutions (“pI variants” or“pI substitutions”) into one or both of the monomers. As shown herein,the separation of the heterodimers from the two homodimers can beaccomplished if the pIs of the two monomers differ by as little as 0.1pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use in thepresent invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components, for example in thetriple F format, the starting pI of the scFv and Fab of interest. Thatis, to determine which monomer to engineer or in which “direction” (e.g.more positive or more negative), the Fv sequences of the two targetantigens are calculated and a decision is made from there. As is knownin the art, different Fvs will have different starting pIs which areexploited in the present invention. In general, as outlined herein, thepIs are engineered to result in a total pI difference of each monomer ofat least about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein. Furthermore, as will be appreciated by those in the art andoutlined herein, in some embodiments, heterodimers can be separated fromhomodimers on the basis of size. As shown in FIG. 2, for example,several of the formats allow separation of heterodimers and homodimerson the basis of size.

A. Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric antibodies in a variety of formats, which utilizeheterodimeric variants to allow for heterodimeric formation and/orpurification away from homodimers. A number of heterodimerizationvariants are shown in FIG. 4.

There are a number of suitable pairs of sets of heterodimerization skewvariants. These variants come in “pairs” of “sets”. That is, one set ofthe pair is incorporated into the first monomer and the other set of thepair is incorporated into the second monomer. It should be noted thatthese sets do not necessarily behave as “knobs in holes” variants, witha one-to-one correspondence between a residue on one monomer and aresidue on the other; that is, these pairs of sets form an interfacebetween the two monomers that encourages heterodimer formation anddiscourages homodimer formation, allowing the percentage of heterodimersthat spontaneously form under biological conditions to be over 90%,rather than the expected 50% (25 homodimer A/A:50% heterodimer A/B:25%homodimer B/B).

B. Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are included in in theFigures.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA—monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which areincorporated expressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the invention.

A list of suitable skew variants is found in FIG. 4 showing some pairsof particular utility in many embodiments. Of particular use in manyembodiments are the pairs of sets including, but not limited to,S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K;T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Qand T366S/L368A/Y407V: T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C: T366W/S354C). In terms of nomenclature, thepair “S364K/E357Q: L368D/K370S” means that one of the monomers has thedouble variant set S364K/E357Q and the other has the double variant setL368D/K370S; as above, the “strandedness” of these pairs depends on thestarting pI.

C. pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 5. As outlinedherein and shown in the figures, these changes are shown relative toIgG1, but all isotypes can be altered this way, as well as isotypehybrids. In the case where the heavy chain constant domain is fromIgG2-4, R133E and R133Q can also be used.

In one embodiment, for example in the bottle opener format, a preferredcombination of pI variants has one monomer (the negative Fab side)comprising 208D/295E/384D/418E/421D variants(N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) and a secondmonomer (the positive scFv side) comprising a positively charged scFvlinker, including (GKPGS)4 (SEQ ID NO: 26209). However, as will beappreciated by those in the art, the first monomer includes a CH1domain, including position 208. Accordingly, in constructs that do notinclude a CH1 domain (for example for heterodimeric Fc fusion proteinsthat do not utilize a CH1 domain on one of the domains, for example in adual scFv format), a preferred negative pI variant Fc set includes295E/384D/418E/421D variants (Q295E/N384D/Q418E/N421D when relative tohuman IgG1).

Accordingly, in some embodiments, one monomer has a set of substitutionsfrom FIG. 5 and the other monomer has a charged linker (either in theform of a charged scFv linker because that monomer comprises an scFv ora charged domain linker, as the format dictates).

1. Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIG. 21 of US Publ. 2014/0370013, hereby incorporated byreference. That is, IgG1 is a common isotype for therapeutic antibodiesfor a variety of reasons, including high effector function. However, theheavy constant region of IgG1 has a higher pI than that of IgG2 (8.10versus 7.31). By introducing IgG2 residues at particular positions intothe IgG1 backbone, the pI of the resulting monomer is lowered (orincreased) and additionally exhibits longer serum half-life. Forexample, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has aglutamic acid (pI 3.22); importing the glutamic acid will affect the pIof the resulting protein. As is described below, a number of amino acidsubstitutions are generally required to significant affect the pI of thevariant antibody. However, it should be noted as discussed below thateven changes in IgG2 molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

D. Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the FIG. 19 of US Pub.2014/0370013. As discussed herein, which monomer to engineer isgenerally decided by the inherent pI of the Fv and scaffold regions.Alternatively, the pI of each monomer can be compared.

E. pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall'Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall'Acqua et al.2002, J. Immunol. 169:5171-5180, entirely incorporated by reference).The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid therelease of the Fc back into the blood. Therefore, the Fc mutations thatwill increase Fc's half-life in vivo will ideally increase FcRn bindingat the lower pH while still allowing release of Fc at higher pH. Theamino acid histidine changes its charge state in the pH range of 6.0 to7.4. Therefore, it is not surprising to find His residues at importantpositions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

F. Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

G. FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto FcγRIIIa results in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell). Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495, 11/538,406,all of which are expressly incorporated herein by reference in theirentirety and specifically for the variants disclosed therein. Particularvariants that find use include, but are not limited to, 236A, 239D,239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 2591, 428L/4345, 2591/308F, 4361/428L, 4361 or V/4345,436V/428L and 2591/308F/428L.

H. Ablation Variants

Similarly, another category of functional variants are “FcγR ablationvariants” or “Fc knock out (FcKO or KO)” variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific immunomodulatoryantibodies desirable to ablate FcγRIIIa binding to eliminate orsignificantly reduce ADCC activity such that one of the Fc domainscomprises one or more Fcγ receptor ablation variants. These ablationvariants are depicted in FIG. 6, and each can be independently andoptionally included or excluded, with preferred aspects utilizingablation variants selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236de1/S239K, E233P/L234V/L235A/G236de1/S267K,E233P/L234V/L235A/G236de1/S239K/A327G,E233P/L234V/L235A/G236de1/S267K/A327G and E233P/L234V/L235A/G236de1. Itshould be noted that the ablation variants referenced herein ablate FcγRbinding but generally not FcRn binding.

I. Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In addition, all of thesevariants can be combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, arealso independently and optionally combined with Fc ablation variants, Fcvariants, FcRn variants, as generally outlined herein.

VII. Useful Formats of the Invention

As will be appreciated by those in the art and discussed more fullybelow, the bispecific heterodimeric antibodies of the present inventioncan take on a wide variety of configurations, as are generally depictedin FIG. 2. Some figures depict “single ended” configurations, wherethere is one type of specificity on one “arm” of the molecule and adifferent specificity on the other “arm”. Other figures depict “dualended” configurations, where there is at least one type of specificityat the “top” of the molecule and one or more different specificities atthe “bottom” of the molecule. Thus, the present invention is directed tonovel immunoglobulin compositions that co-engage a different first and asecond antigen.

As will be appreciated by those in the art, the heterodimeric formats(see FIG. 2) of the invention can have different valencies as well as bebispecific. That is, antibodies of the invention can be bivalent andbispecific, wherein a checkpoint target is bound by one ABD and thecostimulatory target is bound by a second ABD (see for example thebottle opener format which is heterodimeric) or the bispecific mAb whichis homodimeric). The heterodimeric antibodies can also be trivalent andbispecific, wherein the first antigen is bound by two ABDs and thesecond antigen by a second ABD (see for example the Central-scFv formatand the trident format). The heterodimeric antibodies can also bebispecific and tetravalent (such as the Central scFv2 format and theDVD-Ig format).

Again, with the exception of the DVD-Ig format and the central-scFv2format, the antibodies are generally formatted such that theco-stimulatory target is bound monovalently.

A. Bottle Opener Format

One heterodimeric scaffold that finds particular use in the presentinvention is the “triple F” or “bottle opener” scaffold format. In thisembodiment, one heavy chain of the antibody contains a single chain Fv(“scFv”, as defined below) and the other heavy chain is a “regular” Fabformat, comprising a variable heavy chain and a light chain. Thisstructure is sometimes referred to herein as “triple F” format(scFv-Fab-Fc) or the “bottle-opener” format, due to a rough visualsimilarity to a bottle-opener. The two chains are brought together bythe use of amino acid variants in the constant regions (e.g. the Fcdomain, the CH1 domain and/or the hinge region) that promote theformation of heterodimeric antibodies as is described more fully below.

There are several distinct advantages to the present “triple F” format.As is known in the art, antibody analogs relying on two scFv constructsoften have stability and aggregation problems, which can be alleviatedin the present invention by the addition of a “regular” heavy and lightchain pairing. In addition, as opposed to formats that rely on two heavychains and two light chains, there is no issue with the incorrectpairing of heavy and light chains (e.g. heavy 1 pairing with light 2,etc.).

Many of the embodiments outlined herein rely in general on the bottleopener format that comprises a first monomer comprising an scFv,comprising a variable heavy and a variable light domain, covalentlyattached using an scFv linker (charged, in many but not all instances),where the scFv is covalently attached to the N-terminus of a first Fcdomain usually through a domain linker (which, as outlined herein caneither be un-charged or charged and can be exogeneous or endogeneous(e.g. all or part of the native hinge domain). The second monomer of thebottle opener format is a heavy chain, and the composition furthercomprises a light chain.

In addition, the Fc domains of the bottle opener format generallycomprise skew variants (e.g. a set of amino acid substitutions as shownin FIG. 4, with particularly useful skew variants being selected fromthe group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K;L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L,K370S: S364K/E357Q, T366S/L368A/Y407V: T366W andT366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants(including those shown in FIG. 6), optionally charged scFv linkers(including those shown in FIG. 8) and the heavy chain comprises pIvariants (including those shown in FIG. 5).

In some embodiments, the bottle opener format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includebottle opener formats that comprise: a) a first monomer (the “scFvmonomer”) that comprises a charged scFv linker (with the +H sequence ofFIG. 8 being preferred in some embodiments), the skew variantsS364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, andan Fv that binds to one target as outlined herein; b) a second monomer(the “Fab monomer”) that comprises the skew variants L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain, makes up an Fv that binds to a second targetas outlined herein; and c) a light chain. In this particular embodiment,suitable monomer Fv pairs include (Fabs listed first, scFvs second) ICOSX PD-1, ICOS X PD-L1, ICOS X CTLA-4, ICOS X LAG-3, ICOS X TIM-3, ICOS XBTLA, ICOS X TIGIT, TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS, CTLA-4×ICOS,LAG-3×ICOS, TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT, OX40×PD-1, OX40×PD-L1,OX40×CTLA-4, OX40×LAG-3, OX40×TIM-3, OX40×BTLA, TIGIT×OX40, PD-1×OX40,PD-L1×OX40, CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40, BTLA×OX40, GITR×TIGIT,GITR×PD-1, GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3, GITR×BTLA,TIGIT×GITR, PD-1×GITR, PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR, TIM-3×GITR,BTLA×GITR, 4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1, 4-1BB×CTLA-4,4-1BB×LAG-3, 4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB, PD-1×4-1BB,PD-L1×4-1BB, CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB and BTLA×4-1BB.

Accordingly, some embodiments include bottle opener formats thatcomprise: a) a first monomer (the “scFv monomer”) that comprises acharged scFv linker (with the +H sequence of FIG. 8 being preferred insome embodiments), the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and an Fv that binds to one target asoutlined herein; b) a second monomer (the “Fab monomer”) that comprisesthe skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain, makes up an Fv that binds to a second targetas outlined herein; and c) a light chain. In some particular embodimentswith these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the bottle opener format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include bottle opener formats that comprise: a) a firstmonomer (the “scFv monomer”) that comprises a charged scFv linker (withthe +H sequence of FIG. 8 being preferred in some embodiments), the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and an Fvthat binds to a first receptor (either a costimulatory or checkpointreceptor) as outlined herein; b) a second monomer (the “Fab monomer”)that comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and avariable heavy domain that, with the variable light domain, makes up anFv that binds to a second receptor as outlined herein (the other of thecostimulatory or checkpoint receptor); and c) a light chain. In someparticular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

Specifically, FIG. 9 shows some bottle opener “backbone” sequences thatare missing the Fv sequences that can be used in the present invention.That is, Fv sequences for the scFv portion and the Fab portion can beused from any combination of ICOS and PD-1, ICOS and CTLA-4, ICOS andLAG-3, ICOS and TIM-3, ICOS and PD-L1, ICOS and BTLA, ICOS and TIGIT,GITR and TIGIT, GITR and PD-1, GITR and CTLA-4, GITR and LAG-3, GITR andTIM-3, GITR and PD-L1, GITR and BTLA, OX40 and PD-1, OX40 and TIGIT,OX40 and CTLA-4, IC OX40 OS and LAG-3, OX40 and TIM-3, OX40 and PD-L1,OX40 and BTLA, 4-1BB and PD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BBand TIM-3, 4-1BB and PD-L1, TIGIT and 4-1BB and 4-1BB and BTLA, incombination with any or all of backbones 1 to 10, with backbone 1 ofparticular use in these combinations.

For bottle opener backbone 1 from FIG. 9 (optionally including the428L/4345 variants) specific Fv combinations of use in the presentinvention include ICOS and PD-1, ICOS and PD-L1 and ICOS and CTLA-4.

For bottle opener backbone 1 from FIG. 9 (optionally including the428L/4345 variants) specific ABDs that bind human ICOS include, but arenot limited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown inFIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

For bottle opener backbone 1 from FIG. 9 (optionally including the428L/4345 variants), specific ABDs that bind human GITR include, but arenot limited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listedin SEQ ID NO:26282-26290.

For bottle opener backbone 1 from FIG. 9 (optionally including the428L/4345 variants), specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

For bottle opener backbone 1 from FIG. 9 (optionally including the428L/4345 variants), specific ABDs that bind human 4-1BB include, butare not limited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:26262-2671.

For bottle opener backbone 1 from FIG. 9 (optionally including the428L/4345 variants), specific ABDs that bind human PD-L1 include, butare not limited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO:3961-4432.

Specific bottle opener embodiments are outlined below.

B. mAb-Fv Format

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-Fv format. In this embodiment, the format relies onthe use of a C-terminal attachment of an “extra” variable heavy domainto one monomer and the C-terminal attachment of an “extra” variablelight domain to the other monomer, thus forming a third antigen bindingdomain (i.e. an “extra” Fv domain), wherein the Fab portions of the twomonomers bind one checkpoint target and the “extra” Fv domain binds acostimulatory target.

In this embodiment, the first monomer comprises a first heavy chain,comprising a first variable heavy domain and a first constant heavydomain comprising a first Fc domain, with a first variable light domaincovalently attached to the C-terminus of the first Fc domain using adomain linker (vh1-CH1-hinge-CH2-CH3-[optional linker]-vl2). The secondmonomer comprises a second variable heavy domain, a second constantheavy domain comprising a second Fc domain, and a third variable heavydomain covalently attached to the C-terminus of the second Fc domainusing a domain linker (vh1-CH1-hinge-CH2-CH3-[optional linker]-vh2. Thisembodiment further utilizes a common light chain comprising a variablelight domain and a constant light domain, which associates with theheavy chains to form two identical Fabs that include two identical Fvs.The two C-terminally attached variable domains make up the “extra” thirdFv. As for many of the embodiments herein, these constructs include skewvariants, pI variants, ablation variants, additional Fc variants, etc.as desired and described herein. In this embodiment, suitable Fv pairsinclude (Fabs listed first, “extra” Fv listed second) ICOS X PD-1, ICOSX PD-L1, ICOS X CTLA-4, ICOS X LAG-3, ICOS X TIM-3, ICOS X BTLA, ICOS XTIGIT, TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS, CTLA-4×ICOS, LAG-3×ICOS,TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT, OX40×PD-1, OX40×PD-L1, OX40×CTLA-4,OX40×LAG-3, OX40×TIM-3, OX40×BTLA, TIGIT×OX40, PD-1×OX40, PD-L1×OX40,CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40, BTLA×OX40, GITR×TIGIT, GITR×PD-1,GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3, GITR×BTLA, TIGIT×GITR,PD-1×GITR, PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR, TIM-3×GITR, BTLA×GITR,4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1, 4-1BB×CTLA-4, 4-1BB×LAG-3,4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB, PD-1×4-1BB, PD-L1×4-1BB,CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB and BTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figures.

In addition, the Fc domains of the mAb-Fv format comprise skew variants(e.g. a set of amino acid substitutions as shown in FIG. 4, withparticularly useful skew variants being selected from the groupconsisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S:S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants (including those shown inFIG. 6), optionally charged scFv linkers (including those shown in FIG.8) and the heavy chain comprises pI variants (including those shown inFIG. 5).

In some embodiments, the mAb-Fv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includemAb-Fv formats that comprise: a) a first monomer that comprises the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to a first checkpoint inhibitor, and a second variable heavydomain; b) a second monomer that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a first variable heavydomain that, with the first variable light domain, makes up the Fv thatbinds to the first receptor (either a costimulatory receptor or acheckpoint receptor) as outlined herein, and a second variable lightchain, that together with the second variable heavy chain forms an Fv(ABD) that binds a second receptor (e.g. the other of the costimulatoryor checkpoint receptor; and c) a light chain comprising a first variablelight domain and a constant light domain. Of particular use in someembodiments in this format, are (Fab-scFv order) ICOS X PD-1, PD-1×ICOS,PD-L1×ICOS, ICOS×PD-L1, GITR×PD-1, OX40×PD-1 and 4-1BB×PD-1. In someparticular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the mAb-Fv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include mAb-Fv formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domainof the light chain, makes up an Fv that binds to a first checkpointinhibitor, and a second variable heavy domain; b) a second monomer thatcomprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domain,makes up the Fv that binds to the first checkpoint inhibitor as outlinedherein, and a second variable light chain, that together with the secondvariable heavy chain forms an Fv (ABD) that binds a second checkpointinhibitors; and c) a light chain comprising a first variable lightdomain and a constant light domain. Of particular use in someembodiments in this format, are (Fab-scFv order) ICOS X PD-1,ICOS×PD-L1, GITR×PD-1, OX40×PD-1 and 4-1BB×PD-1.

For mAb-Fv sequences that are similar to the mAb-scFv backbone 1(optionally including M428L/N434S) from FIG. 75, specific ABDs that bindhuman ICOS are [ICOS]_H0L0 and [ICOS]_H0.66_L0, as well as those shownin FIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

For mAb-Fv sequences that are similar to the mAb-scFv backbone 1(optionally including M428L/N434S) from FIG. 75, specific ABDs that bindhuman PD-L1 are shown in FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO:3961-4432.

For mAb-Fv sequences that are similar to the mAb-scFv backbone 1(optionally including M428L/N434S) from FIG. 75, specific ABDs that bindhuman GITR are those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

For mAb-Fv sequences that are similar to the mAb-scFv backbone 1(optionally including M428L/N434S) from FIG. 75, specific ABDs that bindhuman 4-1BB are those in FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO:26262-2671.

For mAb-Fv sequences that are similar to the mAb-scFv backbone 1(optionally including M428L/N434S) from FIG. 75, specific ABDs that bindhuman OX40 are those in FIG. 17, FIG. 72 and FIG. 73 and those listed inSEQ ID NO: 26272-26281.

For mAb-Fv sequences that are similar to the mAb-scFv backbone 1(optionally including M428L/N434S) from FIG. 75, specific ABDs that bindhuman PD-L1 from FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

C. mAb-scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-scFv format. In this embodiment, the format relieson the use of a C-terminal attachment of an scFv to one of the monomers,thus forming a third antigen binding domain, wherein the Fab portions ofthe two monomers bind one receptor target and the “extra” scFv domainbinds the other receptor target (generally the monovalently boundcostimulatory receptor).

In this embodiment, the first monomer comprises a first heavy chain(comprising a variable heavy domain and a constant domain), with aC-terminally covalently attached scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain in eitherorientation (vh1-CH1-hinge-CH2-CH3-[optional linker]-vh2-scFv linker-vl2or vh1-CH1-hinge-CH2-CH3-[optional linker]-vl2-scFv linker-vh2). Thisembodiment further utilizes a common light chain comprising a variablelight domain and a constant light domain, which associates with theheavy chains to form two identical Fabs that bind one of the targetreceptors. As for many of the embodiments herein, these constructsinclude skew variants, pI variants, ablation variants, additional Fcvariants, etc. as desired and described herein. In this embodiment,suitable Fv pairs include (Fabs listed first, scFvs second) ICOS X PD-1,ICOS X PD-L1, ICOS X CTLA-4, ICOS X LAG-3, ICOS X TIM-3, ICOS X BTLA,ICOS X TIGIT, TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS, CTLA-4×ICOS,LAG-3×ICOS, TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT, OX40×PD-1, OX40×PD-L1,OX40×CTLA-4, OX40×LAG-3, OX40×TIM-3, OX40×BTLA, TIGIT×OX40, PD-1×OX40,PD-L1×OX40, CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40, BTLA×OX40, GITR×TIGIT,GITR×PD-1, GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3, GITR×BTLA,TIGIT×GITR, PD-1×GITR, PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR, TIM-3×GITR,BTLA×GITR, 4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1, 4-1BB×CTLA-4,4-1BB×LAG-3, 4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB, PD-1×4-1BB,PD-L1×4-1BB, CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB and BTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figures.

In addition, the Fc domains of the mAb-scFv format generally compriseskew variants (e.g. a set of amino acid substitutions as shown in FIG.4, with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S:S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants (including those shown inFIG. 6), optionally charged scFv linkers (including those shown in FIG.8) and the heavy chain comprises pI variants (including those shown inFIG. 5).

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includebottle opener formats that comprise: a) a first monomer that comprisesthe skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to a first receptor, and a scFv that binds to the secondreceptor; b) a second monomer that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a first variable heavydomain that, with the first variable light domain, makes up the Fv thatbinds to the first receptor as outlined herein; and c) a light chaincomprising a first variable light domain and a constant light domain. Insome particular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include mAb-scFv formats that comprise: a) a first monomerthat comprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domainof the light chain, makes up an Fv that binds to a first receptor, and ascFv that binds to the second receptor; b) a second monomer thatcomprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domain,makes up the Fv that binds to the first checkpoint inhibitor as outlinedherein, and c) a light chain comprising a first variable light domainand a constant light domain.

In mAb-scFv backbone 1 (optionally including M428L/N434S) from FIG. 75,specific ABDs that bind human ICOS are shown in FIG. 19, FIG. 20, FIG.24, FIG. 68 and FIG. 77 as well as SEQ ID NO: 27869-28086, 28087-28269,27193-27335, 28549-28556 and 28557-28665.

In mAb-scFv backbone 1 (optionally including M428L/N434S) from FIG. 75,specific ABDs that bind human GITR are those in FIG. 18, FIG. 72 andFIG. 73 and those listed in SEQ ID NO:26282-26290.

In mAb-scFv backbone 1 (optionally including M428L/N434S) from FIG. 75,specific ABDs that bind human 4-1BB include those in FIG. 16, FIG. 72and FIG. 73 and SEQ ID NO: 26262-2671.

In mAb-scFv backbone 1 (optionally including M428L/N434S) from FIG. 75,specific ABDs that bind human OX-40 FIG. 17, FIG. 72 and FIG. 73 andthose listed in SEQ ID NO: 26272-26281.

In mAb-scFv backbone 1 (optionally including M428L/N434S) from FIG. 75,specific ABDs that bind human PD-L1 include FIG. 15, FIG. 73 and FIG. 78and SEQ ID NO: 3961-4432.

D. Central scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the Central-scFv format. In this embodiment, the formatrelies on the use of an inserted scFv domain thus forming a thirdantigen binding domain, wherein the Fab portions of the two monomersbind one receptor target and the “extra” scFv domain binds another(again, generally the costimulatory receptor is bound monovalently). ThescFv domain is inserted between the Fc domain and the CH1-Fv region ofone of the monomers, thus providing a third antigen binding domain.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain (and optional hinge) and Fcdomain, with a scFv comprising a scFv variable light domain, an scFvlinker and a scFv variable heavy domain. The scFv is covalently attachedbetween the C-terminus of the CH1 domain of the heavy constant domainand the N-terminus of the first Fc domain using optional domain linkers(vh1-CH1-[optional linker]-vh2-scFv linker-vl2-[optional linkerincluding the hinge]-CH2-CH3, or the opposite orientation for the scFv,vh1-CH1-[optional linker]-vl2-scFv linker-vh2-[optional linker includingthe hinge]-CH2-CH3). In some embodiments, the optional linker is a hingeor fragment thereof. The other monomer is a standard Fab side (e.g.vh1-CH1-hinge-CH2-CH3). This embodiment further utilizes a common lightchain comprising a variable light domain and a constant light domain,which associates with the heavy chains to form two identical Fabs thatbind a checkpoint inhibitor. As for many of the embodiments herein,these constructs include skew variants, pI variants, ablation variants,additional Fc variants, etc. as desired and described herein. In thisembodiment, suitable Fv pairs include (Fabs listed first, scFvs second)ICOS X PD-1, ICOS X PD-L1, ICOS X CTLA-4, ICOS X LAG-3, ICOS X TIM-3,ICOS X BTLA, ICOS X TIGIT, TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS,CTLA-4×ICOS, LAG-3×ICOS, TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT, OX40×PD-1,OX40×PD-L1, OX40×CTLA-4, OX40×LAG-3, OX40×TIM-3, OX40×BTLA, TIGIT×OX40,PD-1×OX40, PD-L1×OX40, CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40, BTLA×OX40,GITR×TIGIT, GITR×PD-1, GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3,GITR×BTLA, TIGIT×GITR, PD-1×GITR, PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR,TIM-3×GITR, BTLA×GITR, 4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1,4-1BB×CTLA-4, 4-1BB×LAG-3, 4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB,PD-1×4-1BB, PD-L1×4-1BB, CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB andBTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figures.

In addition, the Fc domains of the central scFv format generallycomprise skew variants (e.g. a set of amino acid substitutions as shownin FIG. 4, with particularly useful skew variants being selected fromthe group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K;L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L,K370S: S364K/E357Q, T366S/L368A/Y407V: T366W andT366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants(including those shown in FIG. 6), optionally charged scFv linkers(including those shown in FIG. 8) and the heavy chain comprises pIvariants (including those shown in FIG. 5).

In some embodiments, the central scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includecentral scFv formats that comprise: a) a first monomer that comprisesthe skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to a first receptor; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain, makes up the Fv that binds to thesecond receptor as outlined herein; and c) a light chain comprising afirst variable light domain and a constant light domain. In someparticular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the central scFv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include central-scFv formats that comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a first variable heavy domain that, with the first variable lightdomain of the light chain, makes up an Fv that binds to a first receptorand an scFv domain that binds to a second receptor; b) a second monomerthat comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domain,makes up the Fv that binds to the first checkpoint inhibitor as outlinedherein; and c) a light chain comprising a first variable light domainand a constant light domain. In this embodiment, suitable Fv pairsinclude (Fabs listed first, scFvs second) ICOS X PD-1, PD-1×ICOS, ICOS XPD-L1, PD-L1×ICOS, ICOS X CTLA-4 and CTLA-4×ICOS.

For central-scFv sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind ICOS include, but are not limitedto, shown in FIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well asSEQ ID NO: 27869-28086, 28087-28269, 27193-27335, 28549-28556 and28557-28665.

For central-scFv sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind PD-L1 include, but are not limitedto, those shown in FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO:3961-4432.

For central-scFv sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind GITR include, but are not limitedto, those in FIG. 18, FIG. 72 and FIG. 73 and those listed in SEQ IDNO:26282-26290.

For central-scFv sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind OX40 include, but are not limitedto, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

For central-scFv sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind 4-1BB include, but are not limitedto, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

E. Central-scFv2

One heterodimeric scaffold that finds particular use in the presentinvention is the Central-scFv2 format, which is bispecific andtetravalent. In this embodiment, the format relies on the use of twoinserted scFv domains thus forming third and fourth antigen bindingdomains, wherein the Fab portions of the two monomers bind one receptortarget and the “extra” scFv domains bind another. The scFv domain isinserted between the Fc domain and the CH1-Fv region of the monomers.

In this embodiment, both monomers comprise a first heavy chaincomprising a first variable heavy domain, a CH1 domain (and optionalhinge) and Fc domain, with a scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain. The scFv iscovalently attached between the C-terminus of the CH1 domain of theheavy constant domain and the N-terminus of the first Fc domain usingoptional domain linkers (vh1-CH1-[optionallinker]-vh2-scFvlinker-v12-[optional linker including thehinge]-CH2-CH3, or the opposite orientation for the scFv,vh1-CH1-[optional linker]-vl2-scFv linker-vh2-[optional linker includingthe hinge]-CH2-CH3). In some embodiments, the optional linker is a hingeor fragment thereof. This embodiment further utilizes a common lightchain comprising a variable light domain and a constant light domain,which associates with the heavy chains to form two identical Fabs thatbind a receptor. As for many of the embodiments herein, these constructsinclude skew variants, pI variants, ablation variants, additional Fcvariants, etc. as desired and described herein. In this embodiment,suitable Fv pairs include (Fabs listed first, scFvs second) ICOS X PD-1,ICOS X PD-L1, ICOS X CTLA-4, ICOS X LAG-3, ICOS X TIM-3, ICOS X BTLA,ICOS X TIGIT, TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS, CTLA-4×ICOS,LAG-3×ICOS, TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT, OX40×PD-1, OX40×PD-L1,OX40×CTLA-4, OX40×LAG-3, OX40×TIM-3, OX40×BTLA, TIGIT×OX40, PD-1×OX40,PD-L1×OX40, CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40, BTLA×OX40, GITR×TIGIT,GITR×PD-1, GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3, GITR×BTLA,TIGIT×GITR, PD-1×GITR, PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR, TIM-3×GITR,BTLA×GITR, 4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1, 4-1BB×CTLA-4,4-1BB×LAG-3, 4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB, PD-1×4-1BB,PD-L1×4-1BB, CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB and BTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figures.

In addition, the Fc domains of the central scFv2 format generallycomprise skew variants (e.g. a set of amino acid substitutions as shownin FIG. 4, with particularly useful skew variants being selected fromthe group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K;L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L,K370S: S364K/E357Q, T366S/L368A/Y407V: T366W andT366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants(including those shown in FIG. 6), optionally charged scFv linkers(including those shown in FIG. 8) and the heavy chain comprises pIvariants (including those shown in FIG. 5).

In some embodiments, the central scFv2 format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includecentral scFv formats that comprise: a) a first monomer that comprisesthe skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to a first receptor; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain, makes up the Fv that binds to thesecond receptor as outlined herein; and c) a light chain comprising afirst variable light domain and a constant light domain. In someparticular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the central scFv2 format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include central-scFv formats that comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a first variable heavy domain that, with the first variable lightdomain of the light chain, makes up an Fv that binds to a first receptorand an scFv domain that binds to a second receptor; b) a second monomerthat comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domain,makes up the Fv that binds to the first checkpoint inhibitor as outlinedherein; and c) a light chain comprising a first variable light domainand a constant light domain. In this embodiment, suitable Fv pairsinclude (Fabs listed first, scFvs second) ICOS X PD-1, PD-1×ICOS, ICOS XPD-L1, PD-L1×ICOS, ICOS X CTLA-4 and CTLA-4×ICOS.

For central-scFv2 sequences that utilize the central-scFv2 sequences ofFIGS. 55A-55D suitable Fvs that bind ICOS include, but are not limitedto, shown in FIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well asSEQ ID NO: 27869-28086, 28087-28269, 27193-27335, 28549-28556 and28557-28665.

For central-scFv2 sequences that utilize the central-scFv2 sequences ofFIGS. 55A-55D suitable Fvs that bind PD-L1 include, but are not limitedto, those shown in FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO:3961-4432.

For central-scFv2 sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind GITR include, but are not limitedto, those in FIG. 18, FIG. 72 and FIG. 73 and those listed in SEQ IDNO:26282-26290.

For central-scFv2 sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind OX40 include, but are not limitedto, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

For central-scFv2 sequences that utilize the central-scFv sequences ofFIGS. 55A-55D suitable Fvs that bind 4-1BB include, but are not limitedto, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

F. One Armed mAb

As noted above, surprisingly and unexpectedly, monovalent costimulatoryantibodies comprising a single ABD to the target show efficacy inactivating T cells.

Accordingly, in some embodiments, the invention provides monovalent,monospecific antibodies as shown in Figure FIG. 2N that comprise aheterodimeric Fc domain (for stability). In this embodiment, in thisembodiment, one monomer comprises just an Fc domain, while the othermonomer is a HC (VH1-CH1-hinge-CH2-CH3). This embodiment furtherutilizes a light chain comprising a variable light domain and a constantlight domain, that associates with the heavy chain to form a Fab. As formany of the embodiments herein, these constructs include skew variants,pI variants, ablation variants, additional Fc variants, etc. as desiredand described herein. In this embodiment, suitable ABDs bind acostimulatory receptor such as ICOS, GITR, OX40 or 4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figures.

In addition, the Fc domains of the comprise skew variants (e.g. a set ofamino acid substitutions as shown in FIG. 4, with particularly usefulskew variants being selected from the group consisting of S364K/E357Q:L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E:D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V:T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablationvariants (including those shown in FIG. 6), and the heavy chaincomprises pI variants (including those shown in FIG. 5).

In some embodiments, the one armed scFv-mAb format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments include formats that comprise: a) a first (Fc) monomer thatcomprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K b) a second monomer that comprises theskew variants L368D/K370S, the pI variants Q295E/N384D/Q418E/N421D, theablation variants E233P/L234V/L235A/G236del/S267K, and a first variableheavy domain that, with the first variable light domain, makes up the Fvthat binds to the costimulatory receptor as outlined herein

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

Specific “one armed mAbs” are shown in the Figures and sequence listing.

G. Bispecific mAb

One heterodimeric scaffold that finds particular use in the presentinvention is the bispecific mAb format, which is bispecific andtetravalent. In this embodiment, the format relies on the generation ofseparate homodimeric antibodies which are then recombined. In thisformat, there is one HC-LC pair (VH1-CH1-hinge-CH2-CH3 and VL1-LC) and asecond Hc-LC pair (VH2-CH1-hinge-CH2-CH3 and VL2-LC), e.g. two differentheavy chains and two different light chains. Reference is made toExample 5I(d).

In this format, suitable pairs include ICOS and PD-1, ICOS and CTLA-4,ICOS and LAG-3, ICOS and TIM-3, ICOS and PD-L1, ICOS and BTLA, ICOS andTIGIT, GITR and TIGIT, GITR and PD-1, GITR and CTLA-4, GITR and LAG-3,GITR and TIM-3, GITR and PD-L1, GITR and BTLA, OX40 and PD-1, OX40 andTIGIT, OX40 and CTLA-4, IC OX40 OS and LAG-3, OX40 and TIM-3, OX40 andPD-L1, OX40 and BTLA, 4-1BB and PD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3,4-1BB and TIM-3, 4-1BB and PD-L1, TIGIT and 4-1BB and 4-1BB and BTLA.

In some particular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the ABDcomprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the ABDH3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

H. Central-Fv Format

One heterodimeric scaffold that finds particular use in the presentinvention is the Central-Fv format shown in FIG. 2. In this embodiment,the format relies on the use of an inserted Fv domain thus forming an“extra” third antigen binding domain, wherein the Fab portions of thetwo monomers bind one receptor and the “extra” central-Fv domain bindsanother (generally the costimulatory receptor). The Fv domain isinserted between the Fc domain and the CH1-Fv region of the monomers,thus providing a third antigen binding domain, wherein each monomercontains a component of the Fv (e.g. one monomer comprises a variableheavy domain and the other a variable light domain of the “extra”central Fv domain).

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain, and Fc domain and anadditional variable light domain. The additional variable light domainis covalently attached between the C-terminus of the CH1 domain of theheavy constant domain and the N-terminus of the first Fc domain usingdomain linkers (vh1-CH1-[optional linker]-vl2-hinge-CH2-CH3). The othermonomer comprises a first heavy chain comprising a first variable heavydomain, a CH1 domain and Fc domain and an additional variable heavydomain (vh1-CH1-[optional linker]-vh2-hinge-CH2-CH3). The additionalvariable heavy domain domain is covalently attached between theC-terminus of the CH1 domain of the heavy constant domain and theN-terminus of the first Fc domain using domain linkers. This embodimentutilizes a common light chain comprising a variable light domain and aconstant light domain, that associates with the heavy chains to form twoidentical Fabs that each bind a receptor. The additional variable heavydomain and additional variable light domain form an “extra” central Fvthat binds a second receptor. As for many of the embodiments herein,these constructs include skew variants, pI variants, ablation variants,additional Fc variants, etc. as desired and described herein. In thisembodiment, suitable Fv pairs include (Fabs listed first, “extra”central Fv second) ICOS X PD-1, ICOS X PD-L1, ICOS X CTLA-4, ICOS XLAG-3, ICOS X TIM-3, ICOS X BTLA, ICOS X TIGIT, TIGIT×ICOS, PD-1×ICOS,PD-L1×ICOS, CTLA-4×ICOS, LAG-3×ICOS, TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT,OX40×PD-1, OX40×PD-L1, OX40×CTLA-4, OX40×LAG-3, OX40×TIM-3, OX40×BTLA,TIGIT×OX40, PD-1×OX40, PD-L1×OX40, CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40,BTLA×OX40, GITR×TIGIT, GITR×PD-1, GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3,GITR×TIM-3, GITR×BTLA, TIGIT×GITR, PD-1×GITR, PD-L1×GITR, CTLA-4×GITR,LAG-3×GITR, TIM-3×GITR, BTLA×GITR, 4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1,4-1BB×CTLA-4, 4-1BB×LAG-3, 4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB,PD-1×4-1BB, PD-L1×4-1BB, CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB andBTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figures.

In addition, the Fc domains of the central-Fv format generally compriseskew variants (e.g. a set of amino acid substitutions as shown in FIG.4, with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S:S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants (including those shown inFIG. 6), optionally charged scFv linkers (including those shown in FIG.8) and the heavy chain comprises pI variants (including those shown inFIG. 5).

In some embodiments, the central-Fv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includecentral scFv formats that comprise: a) a first monomer that comprisesthe skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to a first receptor; b) a second monomer that comprises theskew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain, makes up the Fv that binds to thesecond receptor as outlined herein; and c) a light chain comprising afirst variable light domain and a constant light domain. In someparticular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the central-Fv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include central-scFv formats that comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a first variable heavy domain that, with the first variable lightdomain of the light chain, makes up an Fv that binds to a first receptorand an scFv domain that binds to a second receptor; b) a second monomerthat comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domain,makes up the Fv that binds to the first checkpoint inhibitor as outlinedherein; and c) a light chain comprising a first variable light domainand a constant light domain. In this embodiment, suitable Fv pairsinclude (Fabs listed first, scFvs second) ICOS X PD-1, PD-1×ICOS, ICOS XPD-L1, PD-L1×ICOS, ICOS X CTLA-4 and CTLA-4×ICOS.

For central-Fv formats suitable Fvs that bind ICOS include, but are notlimited to, shown in FIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 aswell as SEQ ID NO: 27869-28086, 28087-28269, 27193-27335, 28549-28556and 28557-28665.

For central-Fv formats suitable Fvs that bind PD-L1 include, but are notlimited to, those shown in FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO:3961-4432.

For central-Fv formats suitable Fvs that bind GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

For central-Fv formats suitable Fvs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

For central-Fv formats suitable Fvs that bind 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

For central-FV formats suitable Fvs that bind PD-L1 include, but are notlimited to, those of FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO:3961-4432.

I. One Armed Central-scFv

One heterodimeric scaffold that finds particular use in the presentinvention is the one armed central-scFv format shown in FIG. 1C. In thisembodiment, one monomer comprises just an Fc domain, while the othermonomer includes a Fab domain (a first antigen binding domain), a scFvdomain (a second antigen binding domain) and an Fc domain, where thescFv domain is inserted between the Fc domain and the Fc domain. In thisformat, the Fab portion binds one receptor target and the scFv bindsanother.

In this embodiment, one monomer comprises a first heavy chain comprisinga first variable heavy domain, a CH1 domain and Fc domain, with a scFvcomprising a scFv variable light domain, an scFv linker and a scFvvariable heavy domain. The scFv is covalently attached between theC-terminus of the CH1 domain of the heavy constant domain and theN-terminus of the first Fc domain using domain linkers, in eitherorientation, VH1-CH1-[optional domain linker]-VH2-scFvlinker-VL2-[optional domain linker]-CH2-CH3 or VH1-CH1-[optional domainlinker]-VL2-scFv linker-VH2-[optional domain linker]-CH2-CH3. The secondmonomer comprises an Fc domain (CH2-CH3). This embodiment furtherutilizes a light chain comprising a variable light domain and a constantlight domain, that associates with the heavy chain to form a Fab. As formany of the embodiments herein, these constructs include skew variants,pI variants, ablation variants, additional Fc variants, etc. as desiredand described herein. In this embodiment, suitable Fv pairs include(Fabs listed first, scFvs second) ICOS X PD-1, ICOS X PD-L1, ICOS XCTLA-4, ICOS X LAG-3, ICOS X TIM-3, ICOS X BTLA, ICOS X TIGIT,TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS, CTLA-4×ICOS, LAG-3×ICOS, TIM-3×ICOS,BTLA×ICOS, OX40×TIGIT, OX40×PD-1, OX40×PD-L1, OX40×CTLA-4, OX40×LAG-3,OX40×TIM-3, OX40×BTLA, TIGIT×OX40, PD-1×OX40, PD-L1×OX40, CTLA-4×OX40,LAG-3×OX40, TIM-3×OX40, BTLA×OX40, GITR×TIGIT, GITR×PD-1, GITR×PD-L1,GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3, GITR×BTLA, TIGIT×GITR, PD-1×GITR,PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR, TIM-3×GITR, BTLA×GITR, 4-1BB×TIGIT,4-1BB×PD-1, 4-1BB×PD-L1, 4-1BB×CTLA-4, 4-1BB×LAG-3, 4-1BB×TIM-3,4-1BB×BTLA, TIGIT×4-1BB, PD-1×4-1BB, PD-L1×4-1BB, CTLA-4×4-1BB,LAG-3×4-1BB, TIM-3×4-1BB and BTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figure.

In addition, the Fc domains of the one armed central-scFv formatgenerally comprise skew variants (e.g. a set of amino acid substitutionsas shown in FIG. 4, with particularly useful skew variants beingselected from the group consisting of S364K/E357Q: L368D/K370S;L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K;L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V: T366Wand T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants(including those shown in FIG. 6), optionally charged scFv linkers(including those shown in FIG. 8) and the heavy chain comprises pIvariants (including those shown in FIG. 5).

In some embodiments, the one armed central-scFv format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments include formats that comprise: a) a first monomer thatcomprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, withthe variable light domain of the light chain, makes up an Fv that bindsto a first receptor, and a scFv that binds to the other receptor; b) asecond monomer that comprises the skew variants L368D/K370S, the pIvariants Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, and c) a light chain comprising a firstvariable light domain and a constant light domain. In some particularembodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the one armed central-scFv format includes skewvariants, pI variants, ablation variants and FcRn variants. Accordingly,some embodiments include formats that comprise: a) a first monomer thatcomprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domainof the light chain, makes up an Fv that binds to a first receptor and ascFv domain that binds to a second receptor; b) a second monomer thatcomprises the skew variants L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S. and c) alight chain comprising a first variable light domain and a constantlight domain.

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS] H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

J. One Armed scFv-mAb

One heterodimeric scaffold that finds particular use in the presentinvention is the one armed scFv-mAb format shown in FIG. 2D. In thisembodiment, one monomer comprises just an Fc domain, while the othermonomer uses a scFv domain attached at the N-terminus of the heavychain, generally through the use of a linker: vh1-scFvlinker-vl1-[optional domain linker]-VH2-CH1-hinge-CH2-CH3 or (in theopposite orientation) vl1-scFv linker-vh1-[optional domainlinker]-VH2-CH1-hinge-CH2-CH3. In this format, either the Fab portionbinds one receptor target and the scFv binds another. This embodimentfurther utilizes a light chain comprising a variable light domain and aconstant light domain, that associates with the heavy chain to form aFab. As for many of the embodiments herein, these constructs includeskew variants, pI variants, ablation variants, additional Fc variants,etc. as desired and described herein. In this embodiment, suitable Fvpairs include (Fabs listed first, scFvs second) ICOS X PD-1, ICOS XPD-L1, ICOS X CTLA-4, ICOS X LAG-3, ICOS X TIM-3, ICOS X BTLA, ICOS XTIGIT, TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS, CTLA-4×ICOS, LAG-3×ICOS,TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT, OX40×PD-1, OX40×PD-L1, OX40×CTLA-4,OX40×LAG-3, OX40×TIM-3, OX40×BTLA, TIGIT×OX40, PD-1×OX40, PD-L1×OX40,CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40, BTLA×OX40, GITR×TIGIT, GITR×PD-1,GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3, GITR×BTLA, TIGIT×GITR,PD-1×GITR, PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR, TIM-3×GITR, BTLA×GITR,4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1, 4-1BB×CTLA-4, 4-1BB×LAG-3,4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB, PD-1×4-1BB, PD-L1×4-1BB,CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB and BTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or as shown in the Figures.

In addition, the Fc domains of the comprise skew variants (e.g. a set ofamino acid substitutions as shown in FIG. 4, with particularly usefulskew variants being selected from the group consisting of S364K/E357Q:L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E:D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V:T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablationvariants (including those shown in FIG. 6), optionally charged scFvlinkers (including those shown in FIG. 8) and the heavy chain comprisespI variants (including those shown in FIG. 5).

In some embodiments, the one armed scFv-mAb format includes skewvariants, pI variants, and ablation variants. Accordingly, someembodiments include formats that comprise: a) a first monomer thatcomprises the skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to a first checkpoint inhibitor, and a second variable heavydomain; b) a second monomer that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a first variable heavydomain that, with the first variable light domain, makes up the Fv thatbinds to the first checkpoint inhibitor as outlined herein, and a secondvariable light chain, that together with the second variable heavy chainforms an Fv (ABD) that binds a second checkpoint inhibitors; and c) alight chain comprising a first variable light domain and a constantlight domain. In some particular embodiments with these variants in thisformat:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the one armed scFv-mAb format includes skewvariants, pI variants, ablation variants and FcRn variants. Accordingly,some embodiments include bottle opener formats that comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a first variable heavy domain that, with the first variable lightdomain of the light chain, makes up an Fv that binds to a firstcheckpoint inhibitor, and a second variable heavy domain; b) a secondmonomer that comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domain,makes up the Fv that binds to the first checkpoint inhibitor as outlinedherein, and a second variable light chain, that together with the secondvariable heavy chain forms an Fv (ABD) that binds a second checkpointinhibitors; and c) a light chain comprising a first variable lightdomain and a constant light domain.

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

K. scFv-mAb Format

One heterodimeric scaffold that finds particular use in the presentinvention is the mAb-scFv format shown in FIG. 2E. In this embodiment,the format relies on the use of a N-terminal attachment of a scFv to oneof the monomers, thus forming a third antigen binding domain, whereinthe Fab portions of the two monomers each bind one target and the“extra” scFv domain binds a different target.

In this embodiment, the first monomer comprises a first heavy chain(comprising a variable heavy domain and a constant domain), with aN-terminally covalently attached scFv comprising a scFv variable lightdomain, an scFv linker and a scFv variable heavy domain in eitherorientation ((vh1-scFv linker-vl1-[optional domainlinker]-vh2-CH1-hinge-CH2-CH3) or (with the scFv in the oppositeorientation) ((vl1-scFv linker-vh1-[optional domainlinker]-vh2-CH1-hinge-CH2-CH3)). The second monomer comprises a heavychain VH20CH1-hinge-CH2-CH3. This embodiment further utilizes a commonlight chain comprising a variable light domain and a constant lightdomain, that associates with the heavy chains to form two identical Fabsthat bind one of the target antigens. As for many of the embodimentsherein, these constructs include skew variants, pI variants, ablationvariants, additional Fc variants, etc. as desired and described herein.In this embodiment, suitable Fv pairs include (Fabs listed first, scFvssecond) ICOS X PD-1, ICOS X PD-L1, ICOS X CTLA-4, ICOS X LAG-3, ICOS XTIM-3, ICOS X BTLA, ICOS X TIGIT, TIGIT×ICOS, PD-1×ICOS, PD-L1×ICOS,CTLA-4×ICOS, LAG-3×ICOS, TIM-3×ICOS, BTLA×ICOS, OX40×TIGIT, OX40×PD-1,OX40×PD-L1, OX40×CTLA-4, OX40×LAG-3, OX40×TIM-3, OX40×BTLA, TIGIT×OX40,PD-1×OX40, PD-L1×OX40, CTLA-4×OX40, LAG-3×OX40, TIM-3×OX40, BTLA×OX40,GITR×TIGIT, GITR×PD-1, GITR×PD-L1, GITR×CTLA-4, GITR×LAG-3, GITR×TIM-3,GITR×BTLA, TIGIT×GITR, PD-1×GITR, PD-L1×GITR, CTLA-4×GITR, LAG-3×GITR,TIM-3×GITR, BTLA×GITR, 4-1BB×TIGIT, 4-1BB×PD-1, 4-1BB×PD-L1,4-1BB×CTLA-4, 4-1BB×LAG-3, 4-1BB×TIM-3, 4-1BB×BTLA, TIGIT×4-1BB,PD-1×4-1BB, PD-L1×4-1BB, CTLA-4×4-1BB, LAG-3×4-1BB, TIM-3×4-1BB andBTLA×4-1BB.

The ABD sequences for these combinations can be as disclosed in thesequence listing or in the Figures.

In addition, the Fc domains of the scFv-mAb format comprise skewvariants (e.g. a set of amino acid substitutions as shown in FIG. 4,with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S:S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants (including those shown inFIG. 6), optionally charged scFv linkers (including those shown in FIG.8) and the heavy chain comprises pI variants (including those shown inFIG. 5).

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includebottle opener formats that comprise: a) a first monomer that comprisesthe skew variants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that,with the first variable light domain of the light chain, makes up an Fvthat binds to a first checkpoint inhibitor, and a second variable heavydomain; b) a second monomer that comprises the skew variantsL368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablationvariants E233P/L234V/L235A/G236del/S267K, and a first variable heavydomain that, with the first variable light domain, makes up the Fv thatbinds to the first checkpoint inhibitor as outlined herein, and a secondvariable light chain, that together with the second variable heavy chainforms an Fv (ABD) that binds a second checkpoint inhibitors; and c) alight chain comprising a first variable light domain and a constantlight domain. In some particular embodiments with these variants in thisformat:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS]_H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In some embodiments, the mAb-scFv format includes skew variants, pIvariants, ablation variants and FcRn variants. Accordingly, someembodiments include bottle opener formats that comprise: a) a firstmonomer that comprises the skew variants S364K/E357Q, the ablationvariants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434Sand a first variable heavy domain that, with the first variable lightdomain of the light chain, makes up an Fv that binds to a firstcheckpoint inhibitor, and a second variable heavy domain; b) a secondmonomer that comprises the skew variants L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and afirst variable heavy domain that, with the first variable light domain,makes up the Fv that binds to the first checkpoint inhibitor as outlinedherein, and a second variable light chain, that together with the secondvariable heavy chain forms an Fv (ABD) that binds a second checkpointinhibitors; and c) a light chain comprising a first variable lightdomain and a constant light domain.

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

L. Dual scFv Formats

The present invention also provides dual scFv formats as are known inthe art and shown in FIG. 2B. In this embodiment, the heterodimericbispecific antibody is made up of two scFv-Fc monomers (both in either(vh-scFv linker-v1-[optional domain linker]-CH2-CH3) format or (v1-scFvlinker-vh-[optional domain linker]-CH2-CH3) format, or with one monomerin one orientation and the other in the other orientation.

In this case, all ABDs are in the scFv format, with any combination ofICOS and PD-1, ICOS and CTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS andPD-L1, ICOS and BTLA, GITR and PD-1, GITR and CTLA-4, GITR and LAG-3,GITR and TIM-3, GITR and PD-L1, GITR and BTLA, OX40 and PD-1, OX40 andCTLA-4, OX40 and LAG-3, OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA,4-1BB and PD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3,4-1BB and PD-L1 and 4-1BB and BTLA being useful. The ABD sequences forthese combinations can be as disclosed in the sequence listing or asshown in the Figures.

In addition, the Fc domains of the dual scFv format comprise skewvariants (e.g. a set of amino acid substitutions as shown in FIG. 4,with particularly useful skew variants being selected from the groupconsisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S:S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants (including those shown inFIG. 6), optionally charged scFv linkers (including those shown in FIG.8) and the heavy chain comprises pI variants (including those shown inFIG. 5).

In some embodiments, the dual scFv format includes skew variants, pIvariants, and ablation variants. Accordingly, some embodiments includeformats that comprise: a) a first monomer that comprises the skewvariants S364K/E357Q, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a scFv that binds a first receptor(VH1-scFv linker-VL1-[optional domain linker]-CH2-CH3 or VL1-scFvlinker-VH1-[optional domain linker]-CH2-CH3) and b) a first monomer thatcomprises the skew variants L368D/K370S, the ablation variantsE233P/L234V/L235A/G236del/S267K, and a scFv that binds a first receptor(VH1-scFv linker-VL1-[optional domain linker]-CH2-CH3 or VL1-scFvlinker-VH1-[optional domain linker]-CH2-CH3). pI variants can be asoutlined herein, but most common will be charged scFv linkers ofopposite charge for each monomer. FcRn variants, particularly 428L/434S,can optionally be included. In some particular embodiments with thesevariants in this format:

(1) the format comprises a ABD binds to ICOS that has the ABD of [ICOS]H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the ABDcomprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the ABDH3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1and an Fv binds to GITR;

(6) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1and the Fv binds to OX40;

(7) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1and the ABD binds to ICOS;

8) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1 andan ABD binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

M. Non-Heterodimeric Bispecific Antibodies

As will be appreciated by those in the art, the Fv sequences outlinedherein can also be used in both monospecific antibodies (e.g.“traditional monoclonal antibodies”) or non-heterodimeric bispecificformats (see FIGS. 2J, K and L).

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

Suitable non-heterodimeric bispecific formats are known in the art, andinclude a number of different formats as generally depicted in Spiess etal., Molecular Immunology (67):95-106 (2015) and Kontermann, mAbs 4:2,182-197 (2012), both of which are expressly incorporated by referenceand in particular for the figures, legends and citations to the formatstherein.

N. DVD-Ig Format

In some embodiments, the bispecific antibody is in a “Dual VariableDomain-Ig” or “DVD-Ig™” format (see FIG. 2L) such as is generallydescribed in U.S. Pat. No. 7,612,181, hereby expressly incorporated byreference in its entirety, and in particular for the Figures and Legendstherein. In the DVD-Ig format, the antibody is tetravalent andbispecific, and comprises 4 chains: two homodimeric heavy chains and twoidentical light chains. The heavy chains each have a VH1-(optionallinker)-VH2-CH1-hinge-CH2-CH3 structure and the two light chains eachhave a VL1-optional linker-VL2-CL structure, with VH1 and VL1 forming afirst ABD and the VH2 and VL2 forming a second ABD, where the first andsecond ABDs bind a costimulatory and a checkpoint receptor. In thisembodiment, suitable combinations include ICOS and PD-1, ICOS andCTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS and PD-L1, ICOS and BTLA,GITR and PD-1, GITR and CTLA-4, GITR and LAG-3, GITR and TIM-3, GITR andPD-L1, GITR and BTLA, OX40 and PD-1, OX40 and CTLA-4, OX40 and LAG-3,OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB and PD-1, 4-1BB andCTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB and PD-L1 and 4-1BB andBTLA.

The DVD-Ig™ and Central-scFv2 are two formats that are bispecific andtetravalent, and thus do not bind a costimulatory receptor in amonovalent fashion. Exemplary DVD-Ig™ constructs are shown in FIG. 61.

In some particular embodiments with these variants in this format:

(1) the format comprises the ABD of [ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the ABDcomprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the ABDH3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1and an ABD that binds to GITR;

(6) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1and and ABD that binds to OX40;

(7) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1and an ABD that binds to ICOS;

8) the format comprises the ABD 1G6_L1.194_H1.279 that binds to PD-1 andan ABD that binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

O. Trident Format

In some embodiments, the bispecific antibodies of the invention are inthe “Trident” format as generally described in WO2015/184203, herebyexpressly incorporated by reference in its entirety and in particularfor the Figures, Legends, definitions and sequences of“Heterodimer-Promoting Domains” or “HPDs”, including “K-coil” and“E-coil” sequences. Tridents rely on using two different HPDs thatassociate to form a heterodimeric structure as a component of thestructure, see FIG. 2M. In this embodiment, the Trident format include a“traditional” heavy and light chain (e.g. VH1-CH1-hinge-CH2-CH3 andVL1-CL), a third chain comprising a first “diabody-type binding domain”or “DART®”, VH2-(linker)-VL3-HPD1 and a fourth chain comprising a secondDART®, VH3-(linker)-(linker)-VL2-HPD2. The VH1 and VL1 form a first ABD,the VH2 and VL2 form a second ABD, and the VH3 and VL3 form a third ABD.IN some cases, as is shown in FIG. 2M, the second and third ABDs bindthe same antigen, in this instance generally the checkpoint receptor,e.g. bivalently, with the first ABD binding a costimulatory receptormonovalently. In this embodiment, suitable combinations include ICOS andPD-1, ICOS and CTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS and PD-L1,ICOS and BTLA, GITR and PD-1, GITR and CTLA-4, GITR and LAG-3, GITR andTIM-3, GITR and PD-L1, GITR and BTLA, OX40 and PD-1, OX40 and CTLA-4,OX40 and LAG-3, OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB andPD-1, 4-1BB and CTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB andPD-L1 and 4-1BB and BTLA.

In some particular embodiments with these variants in this format:

(1) the format comprises a Fab ABD binds to ICOS that has the ABD of[ICOS] H0.66_L0;

(2) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvABD comprising the ABD 1G6_L1.194_H1.279 that binds to PD-1;

(3) the format comprises an ICOS ABD of H0.66_L0 combined with the scFvcomprising the ABD H3.23_L0.129 that binds to CTLA-4;

(4) the format comprises an ICOS ABD of H0.66_L0 is combined with theABD 7G8_H.303_L1.34 that binds to LAG-3;

(5) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to GITR;

(6) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to OX40;

(7) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279that binds to PD-1 and the Fab binds to ICOS;

8) the format comprises a scFv comprising the ABD 1G6_L1.194_H1.279 thatbinds to PD-1 and the Fab binds to 4-1BB; and

(9) the format comprises the ABD of H0.66_L0 that binds to ICOS and anABD that binds to PD-L1.

In this format, specific ABDs that bind human ICOS include, but are notlimited to, [ICOS]_H0L0 and [ICOS]H0.66_L0 and and those shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665.

In this format, specific ABDs that bind human GITR include, but are notlimited to, those in FIG. 18, FIG. 72 and FIG. 73 and those listed inSEQ ID NO:26282-26290.

In this format, specific ABDs that bind OX40 include, but are notlimited to, FIG. 17, FIG. 72 and FIG. 73 and those listed in SEQ ID NO:26272-26281.

In this format, specific ABDs that bind human 4-1BB include, but are notlimited to, FIG. 16, FIG. 72 and FIG. 73 and SEQ ID NO: 26262-2671.

In this format, specific ABDs that bind human PD-L1 include, but are notlimited to, FIG. 15, FIG. 73 and FIG. 78 and SEQ ID NO: 3961-4432.

P. Monospecific, Monoclonal Antibodies

As will be appreciated by those in the art, the novel Fv sequencesoutlined herein can also be used in both monospecific antibodies (e.g.“traditional monoclonal antibodies”) or non-heterodimeric bispecificformats. Accordingly, the present invention provides monoclonal(monospecific) antibodies comprising the 6 CDRs and/or the vh and vlsequences from the figures, generally with IgG1, IgG2, IgG3 or IgG4constant regions, with IgG1, IgG2 and IgG4 (including IgG4 constantregions comprising a S228P amino acid substitution) finding particularuse in some embodiments. That is, any sequence herein with a “H_L”designation can be linked to the constant region of a human IgG1antibody.

VIII. Antigen Binding Domains (ABDs) to Target Antigens

The bispecific antibodies of the invention have two different antigenbinding domains (ABDs) that bind to two different target receptorantigens (“target pairs”), in either bivalent, bispecific formats ortrivalent, bispecific formats as generally shown in FIG. 2.

The bispecific antibodies bind to a first target antigen comprising acheckpoint receptor and a second target antigen comprising acostimulatory receptor. Suitable checkpoint receptors as outlined hereininclude PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, BTLA and TIGIT. Suitablecostimulatory receptors as outlined herein include ICOS, GITR, OX40 and4-1BB. As outlined

Suitable target checkpoint antigens include human (and sometimes cyno)PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and BTLA (sequences in the sequencelisting). Accordingly, suitable bispecific antibodies bind ICOS andPD-1, ICOS and CTLA-4, ICOS and LAG-3, ICOS and TIM-3, ICOS and PD-L1,ICOS and BTLA, ICOS and TIGIT, GITR and TIGIT, GITR and PD-1, GITR andCTLA-4, GITR and LAG-3, GITR and TIM-3, GITR and PD-L1, GITR and BTLA,OX40 and PD-1, OX40 and TIGIT, OX40 and CTLA-4, IC OX40 OS and LAG-3,OX40 and TIM-3, OX40 and PD-L1, OX40 and BTLA, 4-1BB and PD-1, 4-1BB andCTLA-4, 4-1BB and LAG-3, 4-1BB and TIM-3, 4-1BB and PD-L1, TIGIT and4-1BB and 4-1BB and BTLA.

Note that generally these bispecific antibodies are named“anti-PD-1×anti-CTLA-4”, or generally simplistically or for ease (andthus interchangeably) as “PD-1×CTLA-4”, etc. for each pair. Note thatunless specified herein, the order of the antigen list in the name doesnot confer structure; that is a PD-1×CTLA-4 bottle opener antibody canhave the scFv bind to PD-1 or CTLA-4, although in some cases, the orderspecifies structure as indicated.

As is more fully outlined herein, these combinations of ABDs can be in avariety of formats, as outlined below, generally in combinations whereone ABD is in a Fab format and the other is in an scFv format. Asdiscussed herein and shown in FIG. 2, some formats use a single Fab anda single scFv (A, C and D), and some formats use two Fabs and a singlescFv (E, F, G, H and I).

A. Antigen Binding Domains

As discussed herein, the bispecific checkpoint heterodimeric antibodiesof the invention include two antigen binding domains (ABDs), each ofwhich bind to a different checkpoint protein. As outlined herein, theseheterodimeric antibodies can be bispecific and bivalent (each antigen isbound by a single ABD, for example, in the format depicted in FIG. 2A),bispecific and trivalent (one antigen is bound by a single ABD and theother is bound by two ABDs, for example as depicted in FIG. 2F, G, H, Ior M), or bispecific and tetravalent (both antigens are bound by twoABDs, for example as depicted in FIGS. 2J and L).

In addition, in general, one of the ABDs comprises a scFv as outlinedherein, in an orientation from N- to C-terminus of vh-scFv linker-vl orvl-scFv linker-vh. One or both of the other ABDs, according to theformat, generally is a Fab, comprising a vh domain on one protein chain(generally as a component of a heavy chain) and a vl on another proteinchain (generally as a component of a light chain). Note that the“trident” format uses DART®s, which are similar to scFvs except theorientation is different and in general the linkers can be slightlylonger.

The invention provides a number of ABDs that bind to a number ofdifferent receptor proteins, as outlined below. As will be appreciatedby those in the art, any set of 6 CDRs or vh and vl domains can be inthe scFv format or in the Fab format, which is then added to the heavyand light constant domains, where the heavy constant domains comprisevariants (including within the CH1 domain as well as the Fc domain). ThescFv sequences contained in the sequence listing utilize a particularcharged linker, but as outlined herein, uncharged or other chargedlinkers can be used, including those depicted in FIG. 8.

In addition, as discussed above, the numbering used in the SequenceListing for the identification of the CDRs is Kabat, however, differentnumbering can be used, which will change the amino acid sequences of theCDRs as shown in Table 1.

For all of the variable heavy and light domains listed herein, furthervariants can be made. As outlined herein, in some embodiments the set of6 CDRs can have from 0, 1, 2, 3, 4 or 5 amino acid modifications (withamino acid substitutions finding particular use), as well as changes inthe framework regions of the variable heavy and light domains, as longas the frameworks (excluding the CDRs) retain at least about 80, 85 or90% identity to a human germline sequence selected from those listed inFIG. 1 of U.S. Pat. No. 7,657,380, which Figure and Legend isincorporated by reference in its entirety herein. Thus, for example, theidentical CDRs as described herein can be combined with differentframework sequences from human germline sequences, as long as theframework regions retain at least 80, 85 or 90% identity to a humangermline sequence selected from those listed in FIG. 1 of U.S. Pat. No.7,657,380. Alternatively, the CDRs can have amino acid modifications(e.g. from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs(that is, the CDRs can be modified as long as the total number ofchanges in the set of 6 CDRs is less than 6 amino acid modifications,with any combination of CDRs being changed; e.g. there may be one changein vlCDR1, two in vhCDR2, none in vhCDR3, etc.)), as well as havingframework region changes, as long as the framework regions retain atleast 80, 85 or 90% identity to a human germline sequence selected fromthose listed in FIG. 1 of U.S. Pat. No. 7,657,380.

B. PD-1 Antigen Binding Domains

In some embodiments, one of the ABDs binds PD-1. Suitable sets of 6 CDRsand/or vh and vl domains, as well as scFv sequences, are depicted inFIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG. 74, FIG. 76 and SEQ IDNO:1-2392, 3125-3144, 4697-7594 and 4697-21810, and include thosesequences in the sequence listing with the identifiers1G6_H1.279_L1.194; 1G6_H1.280_L1.224; 1G6_L1.194_H1.279;1G6_L1.210_H1.288; and 2E9_H1L1.

As will be appreciated by those in the art, suitable anti-PD-1 ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences of these sequences. Suitable ABDs can also include the entirevh and vl sequences as depicted in these sequences and Figures, used asscFvs or as Fabs. In many of the embodiments herein that contain an Fvto PD-1, it is the scFv monomer that binds PD-1. As discussed herein,the other of the target pair when PD-1 is one of the antigens isselected from ICOS (suitable sequences are shown in FIG. 19, FIG. 20,FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO: 27869-28086,28087-28269, 27193-27335, 28549-28556 and 28557-28665 (which can be scFvsequences, CDR sequence sets or vh and vl sequences)), GITR, OX40 and4-1BB.

Particularly useful ABDs that bind human PD-1 include, but are notlimited to, 1G6_H1.279_L1.194, 1G6_H1.280_L1.224; 1G6_L1.194_H1.279,1G6_L1.210_H1.288 and 2E9_H1L1.

Additionally useful vh and vl sequences that bind human PD-1 are shownin FIG. 76.

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to PD-1, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to PD-1, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

Specific preferred embodiments include the 1G6_L1.194_H1.279 anti-PD-1Fv, in a scFv format, included within any of the bottle opener formatbackbones of FIG. 9.

Specific preferred embodiments include the 1G6_L1.194_H1.279 anti-PD-1Fv, in a scFv format, included within any of the format backbones ofFIGS. 55A-55D.

Specific preferred embodiments include the 1G6_L1.194_H1.279 anti-PD-1Fv, in a scFv format, included within any of the mAb-scFv formatbackbones of FIG. 75.

Other embodiments utilize any of the anti-PD-1 vh and vl domain pairs(either as scFvs or Fabs) as shown in FIG. 76 in any format shown inFIG. 2.

C. CTLA-4 Antigen Binding Domains

In some embodiments, one of the ABDs binds CTLA-4. Suitable sets of 6CDRs and/or vh and vl domains, as well as scFv sequences, are depictedin SEQ ID NOs: 2393-2414 and 3737-3816, as well as sequences ofparticular interest in some embodiments are shown in FIG. 12 and FIG. 79and also include those sequences in the sequence listing with theidentifiers [CTLA-4]_H0.25_L0; [CTLA-4]_H0.26_L0; [CTLA-4]_H0.27_L0;[CTLA-4]_H0.29_L0; [CTLA-4]_H0.38_L0; [CTLA-4]_H0.39_L0;0[CTLA-4]_H0.40_L0; [CTLA-4]_H0.70_L0; [CTLA-4]_H0_L0.22;[CTLA-4]_H2_L0; [CTLA-4]_H3.21_L0.124; [CTLA-4]_H3.21_L0.129;[CTLA-4]_H3.21_L0.132; [CTLA-4]_H3.23_L0.124; [CTLA-4]_H3.23_L0.129;[CTLA-4]_H3.23_L0.132; [CTLA-4]_H3.25_L0.124; [CTLA-4]_H3.25_L0.129;[CTLA-4]_H3.25_L0.132; [CTLA-4]_H3.4_L0.118; [CTLA-4]_H3.4_L0.119;[CTLA-4]_H3.4_L0.12; [CTLA-4]_H3.4_L0.121; [CTLA-4]_H3.4_L0.122;[CTLA-4]_H3.4_L0.123; [CTLA-4]_H3.4_L0.124; [CTLA-4]_H3.4_L0.125;[CTLA-4]_H3.4_L0.126; [CTLA-4]_H3.4_L0.127; [CTLA-4]_H3.4_L0.128;[CTLA-4]_H3.4_L0.129; [CTLA-4]_H3.4_L0.130; [CTLA-4]_H3.4_L0.131;[CTLA-4]_H3.4_L0.132; [CTLA-4]_H3.5_L2.1; [CTLA-4]_H3.5_L2.2;[CTLA-4]_H3.5_L2.3; [CTLA-4]_H3_L0; [CTLA-4]_H3_L0.22;[CTLA-4]_H3_L0.44; [CTLA-4]_H3_L0.67; and [CTLA-4]_H3_L0.74.

As will be appreciated by those in the art, suitable anti-CTLA-4 ABDscan comprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences outlined herein. Suitable ABDs can also include the entire vhand vl sequences as depicted in these sequences and Figures, used asscFvs or as Fabs. In many of the embodiments herein that contain an Fvto CTLA-4, it is the scFv monomer that binds CTLA-4.

As discussed herein, the other of the target pair when CTLA-4 is one ofthe antigens is selected from ICOS (suitable sequences are shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665, andthose with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0), GITR, OX40and 4-1BB.

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to CTLA-4, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to CTLA-4, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

D. TIM-3 Antigen Binding Domains

In some embodiments, one of the ABDs binds TIM-3. Suitable sets of 6CDRs and/or vh and vl domains, as well as scFv sequences, are depictedin FIG. 14 and FIG. 81 and SEQ ID NO: 3345-3704, 4585-4696. ABDsequences of particular interest in some embodiments include thosesequences in the sequence listing with the identifiers 1D10_H0L0;1D12_H0L0; 3H3_H1_L2.1; 6C8_H0L0; 6D9_H0_1D12_L0; 7A9_H0L0; 7B11_H0L0;7B11var_H0L0; and 7C2_H0L0.

As will be appreciated by those in the art, suitable anti-TIM-3 ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences of those depicted herein. Suitable ABDs can also include theentire vh and vl sequences as depicted in these sequences and Figures,used as scFvs or as Fabs. In many of the embodiments herein that containan Fv to TIM-3, it is the Fab monomer that binds TIM-3. As discussedherein, the other of the target pair when TIM-3 is one of the antigensis selected ICOS (suitable sequences are shown in FIG. 19, FIG. 20, FIG.24, FIG. 68 and FIG. 77 as well as SEQ ID NO: 27869-28086, 28087-28269,27193-27335, 28549-28556 and 28557-28665, and those with the identifiers[ICOS]_H0.66_L0 and [ICOS]_H0L0), GITR, OX40 and 4-1BB.

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to TIM-3, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to TIM-3, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

LAG-3 Antigen Binding Domains

In some embodiments, one of the ABDs binds LAG-3. Suitable sets of 6CDRs and/or vh and vl domains, as well as scFv sequences, are depictedin FIG. 13, FIG. 80 and in SEQ ID NO: 2415-2604, 3817-3960 also includethose sequences in the sequence listing with the identifiers 2A11_H0L0;2A11_H1.125_L2.113; 2A11_H1.144_L2.142; 2A11_H1_L2.122; 2A11_H1_L2.123;2A11_H1_L2.124; 2A11_H1_L2.25; 2A11_H1_L2.47; 2A11_H1_L2.50;2A11_H1_L2.91; 2A11_H1_L2.93; 2A11_H1_L2.97; 2A11_H1L1; 2A11_H1L2;2A11_H2L2; 2A11_H3L1; 2A11_H3L2; 2A11_H4L1; 2A11_H4L2; 7G8_H0L0;7G8_H1L1; 7G8_H3.18_L1.11; 7G8_H3.23_L1.11; 7G8_H3.28_L1;7G8_H3.28_L1.11; 7G8_H3.28_L1.13; 7G8_H3.30_L1.34; 7G8_H3.30_L1.34; and7G8_H3L1.

As will be appreciated by those in the art, suitable anti-LAG-3 ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences of the sequences herein. Suitable ABDs can also include theentire vh and vl sequences as depicted in these sequences and Figures,used as scFvs or as Fabs. In many of the embodiments herein that containan Fv to LAG-3, it is the Fab monomer that binds LAG-3. As discussedherein, the other of the target pair when LAG-3 is is one of theantigens is selected from ICOS (suitable sequences are shown in FIG. 19,FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO: 27869-28086,28087-28269, 27193-27335, 28549-28556 and 28557-28665, and those withthe identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0), GITR, OX40 and 4-1BB.

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to LAG-3, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to LAG-3, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

E. BTLA Antigen Binding Domains

In some embodiments, one of the ABDs binds BTLA. Suitable sets of 6 CDRsand/or vh and vl domains, as well as scFv sequences, are depicted inFIG. 82, FIG. 84 and SEQ ID NO:3705-3736, and also include thosesequences in the sequence listing with the identifiers 9C6_H0L0;9C6_H1.1_L1; and 9C6_H1.11_L1.

As will be appreciated by those in the art, suitable anti-BTLA ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences depicted herein. Suitable ABDs can also include the entire vhand vl sequences as depicted in these sequences and Figures, used asscFvs or as Fabs. In many of the embodiments herein that contain an Fvto BTLA, it is the Fab monomer that binds BTLA. As discussed herein, theother of the target pair when BTLA is is one of the antigens is selectedfrom ICOS (suitable sequences are shown in FIG. 19, FIG. 20, FIG. 24,FIG. 68 and FIG. 77 as well as SEQ ID NO: 27869-28086, 28087-28269,27193-27335, 28549-28556 and 28557-28665, and those with the identifiers[ICOS]_H0.66_L0 and [ICOS]_H0L0), GITR, OX40 and 4-1BB.

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to BTLA, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to BTLA, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

F. TIGIT Antigen Binding Domains

In some embodiments, one of the ABDs binds TIGIT. Suitable sets of 6CDRs and/or vh and vl domains, as well as scFv sequences, are depictedin FIG. 8? And in SEQ ID NO:4433-4585.

As will be appreciated by those in the art, suitable anti-TIGIT ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences depicted herein. Suitable ABDs can also include the entire vhand vl sequences as depicted in these sequences and Figures, used asscFvs or as Fabs. In many of the embodiments herein that contain an Fvto TIGIT, it is the Fab monomer that binds TIGIT. As discussed herein,the other of the target pair when TIGIT is is one of the antigens isselected ICOS (suitable sequences are shown in FIG. 19, FIG. 20, FIG.24, FIG. 68 and FIG. 77 as well as SEQ ID NO: 27869-28086, 28087-28269,27193-27335, 28549-28556 and 28557-28665, and those with the identifiers[ICOS]_H0.66_L0 and [ICOS]_H0L0), GITR, OX40 and 4-1BB.

G. PD-L1 Antigen Binding Domains

In some embodiments, one of the ABDs binds PD-L1. Suitable sets of 6CDRs and/or vh and vl domains, as well as scFv sequences, are depictedin FIG. 15, FIG. 73 and FIG. 78, and in SEQ ID NO:3961-4432.

As will be appreciated by those in the art, suitable anti-TIGIT ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the vh and vlsequences depicted herein. Suitable ABDs can also include the entire vhand vl sequences as depicted in these sequences and Figures, used asscFvs or as Fabs. In many of the embodiments herein that contain an Fvto TIGIT, it is the Fab monomer that binds TIGIT. As discussed herein,the other of the target pair when TIGIT is is one of the antigens isselected ICOS (suitable sequences are shown in FIG. 19, FIG. 20, FIG.24, FIG. 68 and FIG. 77 as well as SEQ ID NO: 27869-28086, 28087-28269,27193-27335, 28549-28556 and 28557-28665, and those with the identifiers[ICOS]_H0.66_L0 and [ICOS]_H0L0), GITR, OX40 and 4-1BB.

H. ICOS Antigen Binding Domains

In some embodiments, one of the ABDs binds ICOS. Suitable sets of 6 CDRsand/or vh and vl domains, as well as scFv sequences ICOS (suitablesequences are shown in FIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 aswell as SEQ ID NO: 27869-28086, 28087-28269, 27193-27335, 28549-28556and 28557-28665, and those with the identifiers [ICOS]_H0.66_L0 and[ICOS]_H0L0.

As will be appreciated by those in the art, suitable anti-ICOS ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the sequencesdisclosed herein. Suitable ABDs can also include the entire vh and vlsequences as depicted in these sequences and Figures, used as scFvs oras Fabs. In many of the embodiments herein that contain an Fv to ICOS,it is the Fab monomer that binds ICOS. As discussed herein, the other ofthe target pair when ICOS is one of the antigens is selected from PD-1are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG. 74,FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and 4697-21810, andinclude those sequences in the sequence listing with the identifiers1G6_H1.279_L1.194; 1G6_H1.280_L1.224; 1G6_L1.194_H1.279;1G6_L1.210_H1.288; and 2E9_H1L1 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), CTLA-4 (suitable sequences aredepicted in FIGS. 12, 72 and 79, as well as SEQ ID NO:2393-2414 and3737-3816 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), TIM-3 (suitable sequences are depicted in FIG. 14, FIG. 81and SEQ ID NO: 3345-3704, 4585-4696 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), LAG-3 (suitable sequences aredepicted in FIG. 13, FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (whichcan be scFv sequences, CDR sequence sets or vh and vl sequences)), BTLA(suitable sequences are depicted in FIGS. 82 and 84 and SEQ ID NO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), and TIGIT (suitable sequences are depicted in Figure XX andSEQ ID NO:4433-4585 (which can be scFv sequences, CDR sequence sets orvh and vl sequences)).

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to ICOS, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to ICOS, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

Specific preferred embodiments include the [ICOS]_H0.66_L0 anti-ICOS Fv,in a Fab format, included within any of the bottle opener formatbackbones of FIG. 9.

Specific preferred embodiments include the [ICOS]_H0_L0 anti-ICOS Fv, ina scFv format, included within any of the bottle opener format backbonesof FIG. 9.

Specific preferred embodiments include the [ICOS]_H0.66_L0 anti-ICOS Fv,in a scFv format, included within any of the mAb-scFv format backbonesof FIG. 75.

Specific preferred embodiments include the [ICOS]_H0.66_L0 anti-ICOS Fv,in a Fab format, included within any of the mAb-scFv format backbones ofFIG. 75.

Specific preferred embodiments include the [ICOS]_H0.66_L0 anti-ICOS Fv,in a Fab format, included within any of the format backbones of FIGS.55A-55D.

I. GITR Antigen Binding Domains

In some embodiments, one of the ABDs binds GITR. Suitable sets of 6 CDRsand/or vh and vl domains, as well as scFv sequences FITR (suitablesequences are shown in FIG. 18, FIG. 72 and FIG. 73, and in SEQ ID NO:26282-26290.

As will be appreciated by those in the art, suitable anti-GITR ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the sequencesdisclosed herein. Suitable ABDs can also include the entire vh and vlsequences as depicted in these sequences and Figures, used as scFvs oras Fabs. In many of the embodiments herein that contain an Fv to GITR,it is the Fab monomer that binds GITR. As discussed herein, the other ofthe target pair when GITR is one of the antigens is selected from PD-1are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG. 74,FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and 4697-21810, andinclude those sequences in the sequence listing with the identifiers1G6_H1.279_L1.194; 1G6_H1.280_L1.224; 1G6_L1.194_H1.279;1G6_L1.210_H1.288; and 2E9_H1L1 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), CTLA-4 (suitable sequences aredepicted in FIGS. 12, 72 and 79, as well as SEQ ID NO:2393-2414 and3737-3816 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), TIM-3 (suitable sequences are depicted in FIG. 14, FIG. 81and SEQ ID NO: 3345-3704, 4585-4696 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), LAG-3 (suitable sequences aredepicted in FIG. 13, FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (whichcan be scFv sequences, CDR sequence sets or vh and vl sequences)), BTLA(suitable sequences are depicted in FIGS. 82 and 84 and SEQ ID NO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), and TIGIT (suitable sequences are depicted in Figure XX andSEQ ID NO:4433-4585 (which can be scFv sequences, CDR sequence sets orvh and vl sequences)).

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to GITR, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to GITR, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

J. OX40 Antigen Binding Domains

In some embodiments, one of the ABDs binds OX40. Suitable sets of 6 CDRsand/or vh and vl domains, as well as scFv sequences for OX40 areprovided (suitable sequences are shown in FIG. 17, FIG. 72 and FIG. 73,and in SEQ ID NO: 26272-26281.

As will be appreciated by those in the art, suitable anti-OX40 ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the sequencesdisclosed herein. Suitable ABDs can also include the entire vh and vlsequences as depicted in these sequences and Figures, used as scFvs oras Fabs. In many of the embodiments herein that contain an Fv to OX40,it is the Fab monomer that binds GITR. As discussed herein, the other ofthe target pair when OX40 is one of the antigens is selected from PD-1are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG. 74,FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and 4697-21810, andinclude those sequences in the sequence listing with the identifiers1G6_H1.279_L1.194; 1G6_H1.280_L1.224; 1G6_L1.194_H1.279;1G6_L1.210_H1.288; and 2E9_H1L1 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), CTLA-4 (suitable sequences aredepicted in FIGS. 12, 72 and 79, as well as SEQ ID NO:2393-2414 and3737-3816 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), TIM-3 (suitable sequences are depicted in FIG. 14, FIG. 81and SEQ ID NO: 3345-3704, 4585-4696 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), LAG-3 (suitable sequences aredepicted in FIG. 13, FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (whichcan be scFv sequences, CDR sequence sets or vh and vl sequences)), BTLA(suitable sequences are depicted in FIGS. 82 and 84 and SEQ ID NO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), and TIGIT (suitable sequences are depicted in Figure XX andSEQ ID NO:4433-4585 (which can be scFv sequences, CDR sequence sets orvh and vl sequences)).

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to OX40, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to OX40, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

K. 4-1BB Antigen Binding Domains

In some embodiments, one of the ABDs binds 4-1BB. Suitable sets of 6CDRs and/or vh and vl domains, as well as scFv sequences 4-1BB (suitablesequences are shown in FIG. 16, FIG. 72 and FIG. 73, and in SEQ IDNO:26262-2671.

As will be appreciated by those in the art, suitable anti-4-1BB ABDs cancomprise a set of 6 CDRs as depicted in these sequences and Figures,either as they are underlined or, in the case where a differentnumbering scheme is used as described herein and as shown in Table 1, asthe CDRs that are identified using other alignments within the sequencesdisclosed herein. Suitable ABDs can also include the entire vh and vlsequences as depicted in these sequences and Figures, used as scFvs oras Fabs. In many of the embodiments herein that contain an Fv to 4-1BB,it is the Fab monomer that binds 4-1BB. As discussed herein, the otherof the target pair when ICOS is one of the antigens is selected fromPD-1 are depicted in FIG. 3, FIG. 10, FIG. 11, FIG. 72, FIG. 73, FIG.74, FIG. 76 and SEQ ID NO:1-2392, 3125-3144, 4697-7594 and 4697-21810,and include those sequences in the sequence listing with the identifiers1G6_H1.279_L1.194; 1G6_H1.280_L1.224; 1G6_L1.194_H1.279;1G6_L1.210_H1.288; and 2E9_H1L1 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), CTLA-4 (suitable sequences aredepicted in FIGS. 12, 72 and 79, as well as SEQ ID NO:2393-2414 and3737-3816 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), TIM-3 (suitable sequences are depicted in FIG. 14, FIG. 81and SEQ ID NO: 3345-3704, 4585-4696 (which can be scFv sequences, CDRsequence sets or vh and vl sequences)), LAG-3 (suitable sequences aredepicted in FIG. 13, FIG. 80 and SEQ ID NO:2415-2604, 3817-3960 (whichcan be scFv sequences, CDR sequence sets or vh and vl sequences)), BTLA(suitable sequences are depicted in FIGS. 82 and 84 and SEQ ID NO?3705-3736 (which can be scFv sequences, CDR sequence sets or vh and vlsequences)), and TIGIT (suitable sequences are depicted in Figure XX andSEQ ID NO:4433-4585 (which can be scFv sequences, CDR sequence sets orvh and vl sequences)).

In addition to the parental CDR sets disclosed in the sequence listingthat form an ABD to 4-1BB, the invention provides variant CDR sets. Inone embodiment, a set of 6 CDRs can have 1, 2, 3, 4 or 5 amino acidchanges from the parental CDRs, as long as the ABD is still able to bindto the target antigen, as measured at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

In addition to the parental variable heavy and variable light domainsdisclosed herein that form an ABD to 4-1BB, the invention providesvariant vh and vl domains. In one embodiment, the variant vh and vldomains each can have from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from the parental vh and vl domain, as long as the ABD is stillable to bind to the target antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g. Octet assay) assay, with the latter findingparticular use in many embodiments. In another embodiment, the variantvh and vl are at least 90, 95, 97, 98 or 99% identical to the respectiveparental vh or vl, as long as the ABD is still able to bind to thetarget antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g. Octetassay) assay, with the latter finding particular use in manyembodiments.

L. Specific Bispecific Embodiments

The invention provides a number of particular bispecific antibodies asoutlined below.

1. ICOS×PD-1

The invention provides bispecific heterodimeric antibodies that bindICOS and PD-1 each monovalently, and in some cases as outlined herein,both bivalently.

In one embodiment, the PD-1 ABD is 1G6_L1.194_H1.279 and the ICOS ABD isselected from sequences shown in FIG. 19, FIG. 20, FIG. 24, FIG. 68 andFIG. 77 as well as SEQ ID NO: 27869-28086, 28087-28269, 27193-27335,28549-28556 and 28557-28665, and those with the identifiers[ICOS]_H0.66_L0 and [ICOS]_H0L0.

In one embodiment, the ICOS X PD-1 bispecific antibody is selected fromXENP numbers 20261, 20730, 20896, 22432-22438, 22731-22748, 22878-22894,22931-22932, 22950-22961, 23090-23093, 23295-23296, 23301, 23405, 23408,23410 (all without 428L/4345, although they can have thosesubstitutions); XENP numbers 22730, 22917-22928, 22935-22937,22974-22979, 22995-22996, 23001, 23103, 23104 (all with 428L/4345,although they can not have those); XENP numbers 23411, 21828, 21829,21830, 21831, 22348, 23059 (using prior art ICOS sequences); XENPnumbers 18920, 24125, 24130 (additional bottle openers); XENP 23406,23407, 24128 (ICOS×PD-1 central-scFv), XENP24123 (ICOS×PD-1central-scFv2); XENP24134 (ICOS×PD-1 bispecific mAb) 24122 (ICOS×PD-1DVD-Ig), XENP 24132, 24133 (ICOS×PD-1 Trident).

2. ICOS×PD-L1

In this embodiment, the ICOS ABD is selected from sequences shown inFIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665, andthose with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.

3. ICOS×CTLA-4

In this embodiment, the ICOS ABD is selected from sequences shown inFIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665, andthose with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.

In one embodiment, a bottle opener format with a Fab ICOS ABD of[ICOS]H0.66_L0 is paired with a scFv CTLA-4 ABD of[CTLA-4]_H3.32_L0.129, particularly in bottle opener backbone 1 fromFIG. 9.

4. ICOS×LAG-3

In this embodiment, the ICOS ABD is selected from sequences shown inFIG. 19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665, andthose with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.

5. ICOS×TIM-3

I this embodiment, the ICOS ABD is selected from sequences shown in FIG.19, FIG. 20, FIG. 24, FIG. 68 and FIG. 77 as well as SEQ ID NO:27869-28086, 28087-28269, 27193-27335, 28549-28556 and 28557-28665, andthose with the identifiers [ICOS]_H0.66_L0 and [ICOS]_H0L0.

M. Homologous Antibodies

The invention further provides antibodies that share amino acid sequenceidentity with the antibodies outlined herein.

In one embodiment, bispecific antibodies are made that have amino acidvariants in one or more of the CDRs of the Vh and V1 sequences outlinedherein and in the Figures. In one embodiment, antibodies are providedthat have 1, 2, 3, 4 or 5 amino acid differences in one or more of theCDRs of the vh and vl chains outlined herein. These amino acid variantscan be in one CDR or spread out between more than one CDR. These aminoacid variants can also be in one or both of the Fvs of the bispecificantibody; e.g. there can be 2 amino acid variants in CDRs on the ICOS Fvside (generally a Fab but can be a scFv as outlined herein) and one onthe PD-1 side, etc.

Similarly, the invention provides for antibodies that have at least 95,96, 97 98 or 99% amino acid identity to the sequences outlined herein,and particularly in the variable heavy and/or variable light domains.This sequence identity can also be on one Fv or both Fv of thebispecific antibodies. That is, bispecific antibodies are provided thatare 95-99% identical to the variable heavy and/or variable light domainsoutlined in the figures.

IX. Nucleic Acids of the Invention

The invention further provides nucleic acid compositions encoding thebispecific antibodies of the invention (or, in the case of“monospecific” antibodies, nucleic acids encoding those as well).

As will be appreciated by those in the art, the nucleic acidcompositions will depend on the format and scaffold of the heterodimericprotein. Thus, for example, when the format requires three amino acidsequences, three nucleic acid sequences can be incorporated into one ormore expression vectors for expression. Similarly, some formats (e.g.dual scFv formats such as disclosed in FIG. 2) only two nucleic acidsare needed; again, they can be put into one or two expression vectors.Some formats need 4 amino acids (bispecific mAbs).

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the heterodimericantibodies of the invention. Generally, the nucleic acids are operablylinked to any number of regulatory elements (promoters, origin ofreplication, selectable markers, ribosomal binding sites, inducers,etc.). The expression vectors can be extra-chromosomal or integratingvectors.

The nucleic acids and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g. CHO cells), finding use in manyembodiments.

In some embodiments, nucleic acids encoding each monomer and theoptional nucleic acid encoding a light chain, as applicable depending onthe format, are each contained within a single expression vector,generally under different or the same promoter controls. In embodimentsof particular use in the present invention, each of these two or threenucleic acids are contained on a different expression vector. As shownherein and in 62/025,931, hereby incorporated by reference, differentvector ratios can be used to drive heterodimer formation. That is,surprisingly, while the proteins comprise first monomer:secondmonomer:light chains (in the case of many of the embodiments herein thathave three polypeptides comprising the heterodimeric antibody) in a1:1:2 ratio, these are not the ratios that give the best results.

The heterodimeric antibodies of the invention are made by culturing hostcells comprising the expression vector(s) as is well known in the art.Once produced, traditional antibody purification steps are done,including an ion exchange chromotography step. As discussed herein,having the pIs of the two monomers differ by at least 0.5 can allowseparation by ion exchange chromatography or isoelectric focusing, orother methods sensitive to isoelectric point. That is, the inclusion ofpI substitutions that alter the isoelectric point (pI) of each monomerso that such that each monomer has a different pI and the heterodimeralso has a distinct pI, thus facilitating isoelectric purification ofthe “triple F” heterodimer (e.g., anionic exchange columns, cationicexchange columns). These substitutions also aid in the determination andmonitoring of any contaminating dual scFv-Fc and mAb homodimerspost-purification (e.g., IEF gels, cIEF, and analytical IEX columns).

X. Biological and Biochemical Functionality of the HeterodimericImmunomodulatory Antibodies

Generally the bispecific immunomodulatory antibodies of the inventionare administered to patients with cancer, and efficacy is assessed, in anumber of ways as described herein. Thus, while standard assays ofefficacy can be run, such as cancer load, size of tumor, evaluation ofpresence or extent of metastasis, etc., immuno-oncology treatments canbe assessed on the basis of immune status evaluations as well. This canbe done in a number of ways, including both in vitro and in vivo assays.For example, evaluation of changes in immune status (e.g. presence ofICOS+ CD4+ T cells following ipi treatment) along with “old fashioned”measurements such as tumor burden, size, invasiveness, LN involvement,metastasis, etc. can be done. Thus, any or all of the following can beevaluated: the inhibitory effects of PVRIG on CD4⁺ T cell activation orproliferation, CD8⁺ T (CTL) cell activation or proliferation, CD8⁺ Tcell-mediated cytotoxic activity and/or CTL mediated cell depletion, NKcell activity and NK mediated cell depletion, the potentiating effectsof PVRIG on Treg cell differentiation and proliferation and Treg- ormyeloid derived suppressor cell (MDSC)-mediated immunosuppression orimmune tolerance, and/or the effects of PVRIG on proinflammatorycytokine production by immune cells, e.g., IL-2, IFN-γ or TNF-αproduction by T or other immune cells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and 3H-Thymidineincorporation method,

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In general, gene expression assays are done as is known in the art.

In general, protein expression measurements are also similarly done asis known in the art.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, ILS, IL10, IL13 using well knowntechniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs.

Assays to Measure Efficacy

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is known in the art. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of αβ and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-γ production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T cell exhaustion as measured for an exampleby changes in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases αβ and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells. as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g. CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-α,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perforin expression), cytokine production (e.g. IFNγ andTNF), and cell surface receptor expression (e.g. CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal ineither a reference sample or in control samples, for example testsamples that do not contain an anti-PVRIG antibody of the invention.Similarly, increases of at least one-, two-, three-, four- or five-foldas compared to reference or control samples show efficacy.

XI. Treatments

Once made, the compositions of the invention find use in a number ofoncology applications, by treating cancer, generally by inhibiting thesuppression of T cell activation (e.g. T cells are no longer suppressed)with the binding of the bispecific immunomodulatory antibodies of theinvention.

Accordingly, the heterodimeric compositions of the invention find use inthe treatment of these cancers.

XII. Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (as generally outlined inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, buffers, excipients, or stabilizers are nontoxic to recipientsat the dosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MM) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the bispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an bispecific antibody used in the present invention is about 0.1-100mg/kg.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

XIII Examples

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in USPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein.

A. Example 1: TILs from Multiple Cancer Types Co-Express PD-1 and T CellCostimulatory Receptors

To investigate potential associations between PD-1 and various T cellcostimulatory receptors, RNA sequencing data from The Cancer GenomeAtlas project (TCGA) were used for analysis. V2 RSEM data weredownloaded from FireBrowse (http://firebrowse.org/). Analysis wasperformed using R with custom routines. The correlation between theexpression of PD-1 and eight costimulatory receptors is depicted in FIG.1, along with calculated R2 values (square of the Pearson correlationcoefficient). The data show that PD-1 and several costimulatoryreceptors were co-expressed in cancers including bladder, breast, colon,head & neck, kidney, lung-adeno, lung squamous, ovarian, pancreatic,prostate, and melanoma cancer. Notably, expression of ICOS on TILscorrelates better with that of PD-1 than several other costims.

B. Example 2: Immune Checkpoint Antigen Binding Domains

1. 2A: Anti-PD-1 ABDs

Examples of antibodies which bind PD-1 were generated in bivalent IgG1format with E233P/L234V/L235A/G236de1/S267K substitutions, illustrativesequences for which are depicted in FIG. 10. DNA encoding the variableregions was generated by gene synthesis and was inserted into themammalian expression vector pTT5 by the Gibson Assembly method. Heavychain VH genes were inserted via Gibson Assembly into pTT5 encoding thehuman IgG1 constant region with the substitutions mentioned above. Lightchains VL genes were inserted into pTT5 encoding the human C? constantregion. DNA was transfected into HEK293E cells for expression.Additional PD-1 ABDs (including those derived from the above antibodies)were formatted as Fabs and scFvs for use in costim/checkpoint bispecificantibodies, illustrative sequences for which are depicted respectivelyin FIG. 11 and in the sequence listing.

2. 2B: Anti-CTLA-4 ABDs

Antibodies which bind CTLA-4 were generated in bivalent IgG1 format withE233P/L234V/L235A/G236del/S267K substitutions. DNA encoding the variableregions was generated by gene synthesis and was inserted into themammalian expression vector pTT5 by the Gibson Assembly method. Heavychain VH genes were inserted via Gibson Assembly into pTT5 encoding thehuman IgG1 constant region with the substitutions mentioned above. Lightchains VL genes were inserted into pTT5 encoding the human C? constantregion. DNA was transfected into HEK293E cells for expression.Additional CTLA-4 ABDs (including those derived from the aboveantibodies) were formatted as scFvs for use in costim/checkpointbispecific antibodies, illustrative sequences for which are depicted inFIG. 12 and in the sequence listing.

3. 2C: Anti-LAG-3 ABDs

Examples of antibodies which bind LAG-3 were generated in bivalent IgG1format with E233P/L234V/L235A/G236del/S267K substitutions. DNA encodingthe variable regions was generated by gene synthesis and was insertedinto the mammalian expression vector pTT5 by the Gibson Assembly method.Heavy chain VH genes were inserted via Gibson Assembly into pTT5encoding the human IgG1 constant region with the substitutions mentionedabove. Light chains VL genes were inserted into pTT5 encoding the humanC? constant region. DNA was transfected into HEK293E cells forexpression. Additional LAG-3 ABDs (including those derived from theabove antibodies) were formatted as Fabs for use in costim/checkpointbispecific antibodies, illustrative sequences for which are depicted inFIG. 13 and in the sequence listing.

4. 2D: Anti-TIM-3 ABDs

Examples of antibodies which bind TIM-3 were generated in bivalent IgG1format with E233P/L234V/L235A/G236del/S267K substitutions, exemplarysequences for which are depicted in FIG. 14. DNA encoding the variableregions was generated by gene synthesis and was inserted into themammalian expression vector pTT5 by the Gibson Assembly method. Heavychain VH genes were inserted via Gibson Assembly into pTT5 encoding thehuman IgG1 constant region with the substitutions mentioned above. Lightchains VL genes were inserted into pTT5 encoding the human C? constantregion. DNA was transfected into HEK293E cells for expression. The aboveantibodies were formatted as Fabs for use in costim/checkpointbispecific antibodies.

5. 2E: Anti-PD-L1 ABDs

Prototype antibodies which bind PD-L1 were generated in bivalent IgG1format with E233P/L234V/L235A/G236de1/S267K substitutions, exemplarysequences for which are depicted in FIG. 15. DNA encoding the variableregions was generated by gene synthesis and was inserted into themammalian expression vector pTT5 by the Gibson Assembly method. Heavychain VH genes were inserted via Gibson Assembly into pTT5 encoding thehuman IgG1 constant region with the substitutions mentioned above. Lightchains VL genes were inserted into pTT5 encoding the human C? constantregion. DNA was transfected into HEK293E cells for expression. The aboveantibodies were formatted as Fabs and scFvs for use in costim/checkpointbispecific antibodies.

C. Example 3: Costimulatory Receptor Antigen Binding Domains

Prototype costimulatory receptor antibodies which bind ICOS, GITR, OX40,and 4-1BB were generated in bivalent IgG1 format withE233P/L234V/L235A/G236de1/S267K substitutions, sequences for which aredepicted in FIGS. 16-19. DNA encoding the variable regions was generatedby gene synthesis and was inserted into the mammalian expression vectorpTT5 by the Gibson Assembly method. Heavy chain VH genes were insertedvia Gibson Assembly into pTT5 encoding the human IgG1 constant regionwith the substitutions mentioned above. Light chains VL genes wereinserted into pTT5 encoding the human C? constant region. DNA wastransfected into HEK293E cells for expression. The above antibodies wereformatted as Fabs or scFvs for use in costim/checkpoint bispecificantibodies.

D. Example 4: Engineering Anti-ICOS ABD for Stability and Affinity

The parental variable region of an anti-ICOS antibody (XENP16435;depicted in FIG. 19) was engineered for use as a component in anICOS×checkpoint bispecific antibody. A library of Fv variants engineeredto have optimal affinity and stability was constructed by site-directedmutagenesis (QuikChange, Stratagene, Cedar Creek, Tx.) or additionalgene synthesis and subcloning in Fab-His and scFv-His formats andproduced as described below.

1. 4A: Anti-ICOS Fabs

Amino acid sequences for variant anti-ICOS Fabs are listed in FIG. 20(the polyhistidine (His6 (SEQ ID NO: 28666)) tags have been removed fromthe C-terminal of the Fab heavy chains). DNA encoding the two chainsneeded for Fab expression were generated by gene synthesis and weresubcloned using standard molecular biology techniques into theexpression vector pTT5. The Fab heavy chain included a C-terminalpolyhistidine tag. DNA was transfected into HEK293E cells for expressionand resulting proteins were purified from the supernatant using Ni-NTAchromatography. The resultant anti-ICOS Fabs were characterized forstability and affinity.

Differential Scanning Fluorimetry (DSF) experiments were performed usinga Bio-Rad CFX Connect Real-Time PCR Detection System. Proteins weremixed with SYPRO Orange fluorescent dye and diluted to 0.2 mg/mL in PBS.The final concentration of SYPRO Orange was 10×. After an initial 10minute incubation period at 25° C., proteins were heated from 25 to 95°C. using a heating rate of 1° C./min. A fluorescence measurement wastaken every 30 seconds. Melting temperatures (Tm) were calculated usingthe instrument software. The results are shown in FIG. 21.

A series of affinity screens of the anti-ICOS Fabs to human ICOS wereperformed using Octet, a BioLayer Interferometry (BLI)-based method.Experimental steps for Octet generally included the following:Immobilization (capture of ligand or test article onto a biosensor);Association (dipping of ligand- or test article-coated biosensors intowells containing serial dilutions of the corresponding test article orligand); and Dissociation (returning of biosensors to well containingbuffer) in order to determine the monovalent affinity of the testarticles. Specifically, anti-mouse Fc (AMC) biosensors were used tocapture mouse IgG2a Fc fusion of ICOS and dipped into multipleconcentrations of the test articles. The resulting equilibriumdissociation constants (KD), association rates (ka), and dissociationrates (kd) are presented in Figures FIG. 22 and FIG. 23. Bindingaffinities and kinetic rate constants were obtained by analyzing theprocessed data using a 1:1 binding model using ForteBio Octet DataAnalysis software (ForteBio). The data from a two separate experimentsare depicted in FIG. 22A-B. In a further experiment, streptavidin (SA)biosensors were used to capture ICOS-TEV-Fc-Avi and dipped into the testarticles. The data are depicted in FIG. 23. A number of variantanti-ICOS Fabs including XENP22780, XENP22782, XENP22783, and XENP22784had improved stability while maintaining affinity characteristicssimilar to a Fab comprising the parental variable regions (XENP22050).

2. 4B: Anti-ICOS scFvs

Amino acid sequences for anti-ICOS scFvs are listed in FIG. 24 (thepolyhistidine (His6 (SEQ ID NO: 28666)) tags have been removed from theC-terminal of the scFvs). DNA encoding the scFv was generated by genesynthesis and were subcloned using standard molecular biology techniquesinto the expression vector pTT5. The scFv included a C-terminalpolyhistidine tag. DNA was transfected into HEK293E cells for expressionand resulting proteins were purified from the supernatant using Ni-NTAchromatography. The resultant anti-ICOS scFvs were characterized forstability in DSF experiments as described above. The data is depicted inFIG. 25.

E. Example 5: Anti-ICOS×Anti-PD-1 Bispecific Antibodies

1. 5A: Prototype Costim/Checkpoint Bottle-Openers

Schematics for costim/checkpoint bispecific antibody in thebottle-opener format are depicted as FIG. 2A. Prototype bottle-openerswith costimulatory receptor binding Fab arms based on prototypeanti-GITR mAb (XENP16438), anti-OX40 mAb (XENP16437), anti-4-1BB mAb(XENP14410) and anti-ICOS mAb (XENP16435) and exemplary anti-PD-1 scFv(XENP19692) arm were produced to investigate their effect on cytokinesecretion in an SEB-stimulated PBMC assay. Sequences are depicted inFIG. 26. DNA encoding the three chains needed for bottle-openerexpression were generated by gene synthesis and were subcloned usingstandard molecular biology techniques into the expression vector pTT5.DNA transfected into HEK293E cells for expression and resulting proteinswere purified using standard techniques.

a. 5A(a): Prototype Anti-ICOS×Anti-PD-1 Bottle-Opener Enhances CytokineSecretion

Staphylococcal Enterotoxin B (SEB) is a superantigen that causes T cellactivation and proliferation in a manner similar to that achieved byactivation via the T cell receptor (TCR). Stimulating human PBMC withSEB is a common method for assaying T cell activation and proliferation.PBMCs were simulated with 100 ng/mL SEB for 2 days. Cells were washedtwice and restimulated with 100 ng/mL SEB in combination with 20 μg/mLof the indicated test articles. A first bivalent anti-PD-1 antibodybased on nivolumab (XENP16432), a second bivalent anti-PD-1 mAb(XENP19686), and a bivalent anti-RSV mAb (XENP15074) were used ascontrols. 24 hours after treatment, supernatants were assayed for IL-2.The data depicted in FIG. 27 show that each of the costim/checkpointbottle-openers enhanced cytokine secretion in comparison to the bivalentanti-RSV antibody. Surprisingly, induction of cytokine secretion byXENP22730 is vastly superior to cytokine secretion by bivalent anti-PD-1antibody alone as well as the other prototype costim/checkpointbottle-openers, indicating that addition of ICOS binding enhancescytokine production and that ICOS is a better PD-1 partner than othercostimulatory receptors for a bispecific antibody.

In another experiment, PBMCs were stimulated with 0.01 μg/mL SEB for 3days with 20 μg/mL of indicated test articles. As control, test articleswere also incubated with naive (non-SEB stimulated) PBMCs. 3 days aftertreatment, supernatant was assessed for IL-2 secretion as an indicatorof T cell activation (depicted in FIG. 28). The data show that neitherthe anti-PD-1 bivalent antibody nor the anti-ICOS×anti-PD-1bottle-opener stimulate IL-2 secretion in naive cells. Further, the datashow again that XENP20896 enhances IL-2 secretion more than the bivalentanti-PD-1 antibody alone does and that XENP20896 enhances IL-2 secretionmore than combination of bivalents (XENP16432 and XENP16435) as well ascombination of one-arms (XENP20111 and XENP20266) do.

Costimulatory receptors such as ICOS have previously been found toinduce cytokine production only following crosslinking by bivalentantibodies or multimerized ligands (Viera et al. 2004; Sanmamed et al.2015). In view of the crosslinking mechanisms, it is surprising thatmonovalent ICOS binding by a single arm in a costim×checkpoint blockadebispecific antibody was able to enhance cytokine production. Further, itis notable that the bispecific antibody was able to enhance cytokinesecretion more than bivalent anti-PD-1 mAb in combination with bivalentanti-ICOS mAb (XENP16432+XENP16435).

b. 5A(b): PD-1 and ICOS Double-Positive Cells are Selectively Occupiedby Prototype Anti-ICOS×Anti-PD-1 Bottle-Opener

Selective targeting of tumor-reactive TILs co-expressing immunecheckpoint receptors (e.g. PD-1) and costimulatory receptors as shown inExample 1 over non-tumor reactive T cells expressing immune checkpointreceptors or costimulatory receptors alone could enhance anti-tumoractivity while avoiding peripheral toxicity (as depicted in FIG. 29).

An SEB-stimulated PBMC assay was used to investigate binding ofanti-ICOS×anti-PD-1 bottle-opener to T cells. PBMCs were stimulated with100 ng/mL SEB (staphylococcal enterotoxin B) for 3 days, after which thePBMCs were treated with the indicated test articles for 30 minutes at 4°C. PBMCs were then incubated with APC-labeled one-arm anti-ICOS antibodyand FITC-labeled one-arm anti-PD-1 antibody for 30 minutes at 4° C. FIG.30 and FIG. 31 shows receptor occupancy of a prototypeanti-ICOS×anti-PD-1 bottle-opener (XENP20896), one-arm anti-ICOSantibody (XENP20266) and one-arm anti-PD-1 antibody (XENP20111).

The data show that double-positive cells (expressing both PD-1 and ICOS)are selectively occupied by the anti-ICOS×anti-PD-1 bottle-opener(XENP20896) as depicted in FIG. 30, indicating that monovalent ICOS andPD-1 binding is useful for selective targeting. Further,anti-ICOS×anti-PD-1 bottle-opener (e.g. XENP20896) binds more potentlyto double-positive cells than monovalent, monospecific one-arm anti-PD-1and anti-ICOS as shown in FIG. 31A.

c. 5A(c): Prototype Anti-ICOS×Anti-PD-1 Bottle-Opener EnhanceEngraftment in a GVHD Mouse Study

The prototype anti-ICOS×anti-PD-1 bottle-opener was evaluated in aGraft-versus-Host Disease (GVHD) model conducted in NSG (NOD-SCID-gamma)immunodeficient mice. The mice were engrafted with human PBMCs. When NSGmice are injected with human PBMCs, they develop an autoimmune responseagainst the human PBMCs. Treatment of NSG mice injected with human PBMCswith immune checkpoint antibodies (e.g. anti-PD-1) enhance engraftment.Thus, increased engraftment shows efficacy of the antibodies.

10 million human PBMCs were engrafted into NSG mice via IV-OSP on Day 0followed by dosing with the indicated test articles on Day 1, 8, 15, and22. Human CD45+ cell counts were measured on Day 14 as an indicator ofdisease.

The data depicted in Figure A show that the anti-ICOS×anti-PD-1bottle-opener enhance proliferation of CD45+ cells in humanPBMC-engrafted NSG mice as compared to controls (PBS and PBS+PBMC).Further, enhancement is greater using antibodies of the invention thanthat seen with bivalent anti-PD-1 antibody.

2. 5B: Production of Variant Anti-ICOS×Anti-PD-1 Bottle-Openers withOptimized Anti-ICOS Fab Arms

Variant anti-ICOS×anti-PD-1 bottle-openers comprising anti-ICOS Fabsengineered as described in Example 4A were produced as generallydescribed above. Amino acid sequences for variant anti-ICOS×anti-PD-1bottle-openers are listed in FIG. 32. Amino acid sequences for variantanti-ICOS×anti-PD-1 with FcRn pH 6.0 affinity enhancing substitutionsare listed in FIG. 33.

The resultant anti-ICOS×anti-PD-1 bispecific antibodies werecharacterized for affinity to human and cynomolgus ICOS using Octet asgenerally described above. In a first set of experiments, AMC biosensorswere used to capture mouse IgG2a Fc fusion of ICOS and dipped intomultiple concentrations of the test articles (data depicted in FIG. 34).In further experiments, streptavidin (SA or SAX) biosensors were used tocapture biotinylated human and cynomolgus IgG1 Fc fusions of human andcynomolgus ICOS and dipped into multiple concentrations of the testarticles (data depicted in FIG. 35).

3. 5C: T Cell Surface Binding of Variant Anti-ICOS×Anti-PD-1Bottle-Openers

Binding of anti-ICOS×anti-PD-1 bispecifics to T cells was measured in anSEB-stimulated PBMC assay. Human PBMCs were stimulated with 100 ng/mLSEB for 3 days. PBMCs were then treated with the indicated test articlesand incubated at 4° C. for 30 minutes. After treatment, cells wereincubated with FITC-labeled anti-CD3 antibody and APC-labeled anti-humanIgG Fc secondary antibody. MFI on CD3+ cells are depicted in FIG. 36A-B.

4. 5D: Receptor Occupancy of Variant Anti-ICOS×Anti-PD-1 Bottle-Openerson T Cells

Receptor occupancy of variant anti-ICOS×anti-PD-1 bottle-openers on Tcells was measured in an SEB-stimulated PBMC assay. PBMCs werestimulated with 100 ng/mL SEB (staphylococcal enterotoxin B) for 3 days,after which the PBMCs were treated with the indicated test articles for30 minutes at 4° C. PBMCs were then incubated with APC-labeled one-armanti-ICOS antibody and FITC-labeled one-arm anti-PD-1 antibody for 30minutes at 4° C. FIG. 37 depicts the receptor occupancy of variantanti-ICOS×anti-PD-1 bispecific antibodies, corresponding one-armanti-ICOS antibodies and one-arm anti-PD-1 antibody (XENP20111) on PD-1and ICOS double-positive T cells. Consistent with the prototypeantibodies investigated in Example 1A, each of the bottle-openers bindsmore potently to double-positive cells than monovalent, monospecificone-arm anti-PD-1 and one-arm anti-ICOS antibodies.

5. 5E: In Vitro Activity of Variant Anti-ICOS×Anti-PD-1 Bottle-Openersin a Cytokine Release Assay

Human PBMCs were stimulated with 100 ng/mL SEB for 2 days. Cells werewashed and stimulated again with 100 ng/mL SEB in combination with 20μg/mL of indicated test articles. 24 hours after treatment, cells wereassayed for IL-2 (FIG. 38A) and IFN? (FIG. 38B). The data show thatanti-ICOS×anti-PD-1 bottle-openers stimulated significantly morecytokine release than bivalent anti-PD-1 antibody (XENP16432) alone,bivalent anti-ICOS antibody (XENP16435) alone, or bivalent anti-PD-1antibody plus bivalent anti-ICOS antibody in combination.

In a further experiment, human PBMCs were stimulated with 100 ng/mL SEBfor 2 days. Cells were washed and stimulated again with 100 ng/mL SEB incombination with 20 μg/mL of indicated test articles. 24 hours aftertreatment, cells were assayed for IL-2 (FIG. 39A) and IFN? (FIG. 39B).

6. 5F: In Vivo Activity of Variant Anti-ICOS×Anti-PD-1 Bottle Openers ina GVHD Mouse Study

In a first study, 10 million human PBMCs were engrafted into NSG micevia IV-OSP on Day 0 followed by dosing with the indicated test articleson Day 1. IFN? levels and human CD45+, CD8+ T cell and CD4+ T cellcounts were measured on Day 7, 11 and 14. FIGS. 40A-B respectivelydepicts IFN? levels on Day 7 and 11. FIGS. 41A-B respectively depictCD45+ cell counts on Day 11 and 14. FIGS. 42A-B respectively depict CD8+T cell and CD4+ T cell counts on Day 14. FIG. 43 depicts the change inbody weight in the mice by Day 14 resulting from exacerbation of GVHDdue to T cell expansion and IFN? production.

In a further study with additional variant anti-ICOS×anti-PD-1bottle-openers, 10 million human PBMCs were engrafted into NSG mice viaIV-OSP on Day 0 followed by dosing with the indicated test articles atindicated concentrations on Day 1. IFNγ levels and human CD45+, CD8+ Tcell and CD4+ T cell counts were measured on Day 7 and 14. FIG. 44A-Brespectively depicts IFN? levels on Day 7 and 14. FIG. 45 depicts CD45+cell count on Day 14. FIG. 46A-C respectively depict CD8+ T cell count,CD4+ T cell counts and CD8+/CD4+ ratio on Day 14. Body weight of micewere also measured on Day 12 and 15 and depicted respectively in FIG.47A-B as percentage of initial body weight.

The Figures show that the anti-ICOS×anti-PD-1 bottle-openers enhanceengraftment (as indicated by the proliferation of CD45+ cells, CD8+ Tcells and CD4+ T cells and decrease in body weight of mice). Theobserved activity is correlated to the in vitro potency of each variant.Further, a number of the bottle-openers including XENP20896, XENP22744,XENP23092, XENP22730, XENP23104, XENP22974, and XENP23411 enhanceengraftment much more than the control bivalent anti-PD-1 antibody(XENP16432) does.

7. 5G: Additional Anti-ICOS ABDs Work in Anti-ICOS×Anti-PD-1 BottleOpener

Additional anti-ICOS×anti-PD-1 bottle-openers were produced comprisinganti-ICOS Fabs based on other anti-ICOS ABDs described in Example 3 asgenerally described above. Sequences for the additional bottle-openersare depicted in FIG. 48.

In a first experiment, PBMCs were stimulated with 100 ng/mL SEB for 2days. Cells were washed twice and restimulated with 100 ng/mL SEB incombination with 20 μg/mL of indicated test articles (PBS as control).24 hours after treatment, supernatants were assayed for IL-2concentration as depicted in FIG. 49. In a second experiment, PBMCs werestimulated with 10 ng/mL SEB and treated with 20 μg/mL of indicated testarticles for 3 days (bivalent anti-PD-1 XENP16432 as control).Supernatants were collected and assays for IL-2. FIG. 50 depicts thefold induction in IL-2 over bivalent anti-RSV mAb.

Consistent with the data in Example 5A(a), XENP20896 stimulatedsecretion of IL-2. Notably, the additional bottle-openers comprisingalternative anti-ICOS ABDs were also able to stimulate secretion of IL-2demonstrating that the enhancement of cytokine secretion by ananti-ICOS×anti-PD-1 bottle-opener is not unique to ABDs derived from theparental anti-ICOS ABD described in Example 4.

8. 5H: Clear ICOS Signature is Exhibited by Anti-ICOS×Anti-PD-1Bottle-Openers

a. 5H(a): Anti-ICOS×Anti-PD-1 Bispecific Antibodies Induce AKTPhosphorylation

ICOS ligation induces AKT phosphorylation in activated T cells (Fos, Cet al. 2008), and as such, AKT phosphorylation would be an indicator ofICOS agonism by anti-ICOS×anti-PD-1 bispecific antibodies of theinvention.

Human PBMCs were stimulated with 100 ng/mL SEB for 2 days. Followingstimulation, CD3+ cells were isolated by negative selection usingEasySep™ Human T Cell Enrichment Kit (STEMCELL Technologies, Vancouver,Canada) and then treated with indicated test articles in combinationwith plate bound anti-CD3 antibody (OKT3; 500 ng/mL). Cells were lysed30 minutes after treatment and assayed for total AKT and phosphorylatedAKT (Ser473) by a multiplexed phosphoprotein assay on MULTI-SPOT384-Well Spot plates (Meso Scale Discovery, Rockville, Md.). The dataare depicted in FIG. 51 as percentage of AKT phosphorylated followingtreatment.

The data shows no increase in AKT phosphorylation following treatmentwith negative control bivalent anti-PD-1 mAb (XENP16432). Both bivalentanti-ICOS mAb alone (XENP16435) and XENP16435 in combination withXENP16432 increase AKT phosphorylation demonstrating ICOS ligation andagonism by XENP16435. Surprisingly, despite only monovalent engagementof ICOS, treatment with the anti-ICOS×anti-PD-1 bispecific antibodiesinduces more AKT phosphorylation than treatment with XENP16435 alone andXENP16435 in combination with XENP16432. The positive AKTphosphorylation data demonstrate a clear signature of ICOS activity withthe bispecific antibodies despite monovalent engagement of ICOS.

b. 5H(b): Activated T Helper Cell-Associated Genes Upregulated byAnti-ICOS×Anti-PD-1 Bottle-Openers

Guedan et al. (2014) describes gene expression profiles for activated Thelper cells (i.e. Th1, Th2, and Th17) and regulatory T cells (Tregs)following activation of ICOS signaling domain-based CAR-Ts. They foundthat genes related to activated T helper cells such as IL-17A, IL22,IFN?, TNF, and IL-13 were upregulated, while Treg-related genes such asTGFβ1, SMAD3 and FOXP3 were either unchanged or downregulated.

To investigate if engagement of T cells with anti-ICOS×anti-PD-1bispecific antibodies of the invention led to a similar signature, weused Nanostring technology. PBMCs were stimulated with 100 ng/mL SEB for2 days. Cells were washed 2 times and restimulated with 100 ng/mL SEBand treated with indicated test articles for 24 hours. RNA was extractedfrom cells and assayed by nCounter® PanCancer Immune Profiling Panel(NanoString Technologies, Seattle, Wash.) which assays 770 target genescovering immune response. FIG. 52 depicts mean fold induction inexpression of a number of Th-related and Treg-related genes overbivalent anti-RSV antibody. FIGS. 53A-F respectively depict foldinduction of IL-17A, IL-17F, IL-22, IL-10, IL-9 and IFN? gene expressionby the indicated test articles over induction by bivalent anti-RSV mAb.

As depicted in FIGS. 52-53 and consistent with the observation by Guedenet al., Th-related genes associated with ICOS signaling such as IL-17A,IL-17F, IL-22, IL-9, and IFN? are upregulated following treatment withbivalent anti-ICOS mAb and combination of anti-ICOS and anti-PD-1 mAbs.Notably, expression of Th-related genes associated with ICOS signalingare further upregulated following treatment with an anti-ICOS×anti-PD-1antibody, again indicating a clear ICOS costimulatory signature anddramatic synergy of anti-ICOS and anti-PD-1 engagement by a bispecificantibody.

9. 5I: Alternative Format Anti-ICOS×Anti-PD-1 Bispecific Antibodies

Alternative format costim/checkpoint bispecific antibodies were producedto investigate whether the effect of anti-ICOS×anti-PD-1 bottle-openerswas unique to the bottle-opener format or broadly applicable toanti-ICOS×anti-PD-1 bispecific antibodies.

a. 5I(a): Anti-PD-1×Anti-ICOS Bottle-Opener

Anti-PD-1×anti-ICOS bottle-openers with an anti-PD-1 Fab generated usingDNA encoding anti-PD-1 mAbs (e.g. XENP16432 and XENP29120) as describedin Example 2A and anti-ICOS scFvs as described in Example 4B.Bottle-openers were produced as generally described in Example 5A.Sequences for exemplary anti-PD-1×anti-ICOS bottle-openers are depictedin FIG. 56.

b. 5I(b): Central-scFv

Schematics for the central-scFv format are depicted as FIGS. 55A-55B.DNA encoding anti-ICOS Fab-Fc heavy chains was generated using DNAencoding anti-ICOS Fabs described in Example 4A by standard subcloninginto the expression vector pTT5. DNA encoding anti-ICOS-anti-PD-1Fab-scFv-Fc heavy chains was generated using DNA encoding anti-ICOS Fabsdescribed in Example 4A and anti-PD-1 scFv described in Example 2A by acombination of gene synthesis and standard subcloning into theexpression vector pTT5. DNA was transfected into HEK293E cells forexpression. Sequences for exemplary anti-ICOS×anti-PD-1 central-scFvantibodies are depicted in FIG. 57.

c. 5I(c): Central-scFv2

A schematic for the central-scFv2 format is depicted as FIG. 55C. DNAencoding anti-ICOS-anti-PD-1 Fab-scFv-Fc heavy chains was generatedusing DNA encoding anti-ICOS Fabs described in Example 4A and anti-PD-1scFv described in Example 2A by a combination of gene synthesis andstandard subcloning into the expression vector pTT5. DNA was transfectedinto HEK293E cells for expression. Sequences for exemplaryanti-ICOS×anti-PD-1 central-scFv2 antibodies are depicted in FIG. 58.

d. 5I(d): Bispecific mAb

A schematic for the bispecific mAb format is depicted as FIG. 2K. DNAencoding the anti-ICOS heavy and light chains were generated based onthe DNA encoding the anti-ICOS Fabs described in Example, and DNAencoding anti-PD-1 heavy and light chains were generated based on theDNA encoding the anti-PD-1 mAbs described in Example 2A. Heavy chain VHgenes were inserted via Gibson assembly into pTT5 encoding the humanIgG1 constant region with E233P/L234V/L235A/G236de1/S267K substitutions.Light chain VL genes were inserted into pTT5 encoding the human C?constant region.

DNA was transfected into HEK293E cells for expression as 2 separateantibodies which are separately expressed and purified by Protein Aaffinity (GE Healthcare). These antibodies containheterodimerization-skewing substitutions in the CH3:CH3 interface.2-Mercaptoethylamine-HCl (2-MEA) was used to induce controlled reductionof interchain disulfide bonds in the two parental IgGs. 2-MEA was thenremoved allowing the reoxidation of interchain disulfide bonds to occurenabling recombination of the HC-LC pairs (driven by theheterodimerization mutations). Finally, the trispecific antibodies werepurified by cation exchange chromatography. Such methods are common inthe art (see, for instance, Labrijn (2013) PNAS 110(13):5145-50 or Stropet al. (2012) J Mol Biol 420(3):204-19). Other methods of design andpurification known in the art may be used facilitate bispecific mAbproduction, for instance, common light chain antibodies (Merchant (1998)Nat Biotechnol 16(7):677-81) or heterodimeric Fab domains (Lewis et al.(2014) Nat Biotechnol 32(2):191-8). Sequence for an exemplaryanti-PD-1×anti-ICOS bispecific mAb is depicted in FIG. 59.

e. 5I(e): DVD-IgG

A schematic for the DVD-IgG format is depicted as FIGS. 55A-55D. DNAencoding anti-PD-1-anti-ICOS VH—VH—CH1-Fc heavy chains and VL-VL-C?light chains was generated using DNA encoding anti-ICOS Fabs describedin Example 4A and anti-PD-1 scFv described in Example 2A by acombination of gene synthesis and standard subcloning into theexpression vector pTT5. DNA was transfected into HEK293E cells forexpression. Sequences for exemplary anti-ICOS×anti-PD-1 DVD-IgGs aredepicted in FIG. 60.

f. 5I(f): Trident

A schematic for the Trident format is depicted as FIGS. 55A-55D. DNAencoding anti-PD-1 VL-VH-Fc heavy chains and VL-VH light chains wasgenerated using DNA encoding anti-ICOS Fabs described in Example 4A andanti-PD-1 scFv described in Example 2A by a combination of genesynthesis and standard subcloning into the expression vector pTT5. DNAwas transfected into HEK293E cells for expression. Sequences forexemplary anti-ICOS×anti-PD-1 Tridents are depicted in FIG. 61.

g. 5I(g): Alternative Format Anti-ICOS×Anti-PD-1 Bispecific AntibodiesEnhance Cytokine Secretion

Human PBMCs were stimulated with 200 ng/mL SEB for 2 days. Cells werewashed twice then re-stimulated with 100 ng/mL SEB and treated with theindicated concentrations of the indicated test articles. 24 hours aftertreatment, supernatants were assayed for IL-2. The data are depicted inFIG. 62 and show that each of the alternative format bispecificantibodies enhances IL-2 secretion in comparison to bivalent anti-RSV(XENP15074) control. Notably, the majority of these alternative formatantibodies enhance IL-2 secretion in comparison to a bivalent anti-PD-1antibody.

F. Example 6: Costim/Checkpoint Bispecific Antibodies TargetingDifferent Immune Checkpoint Antigens

1. 6A: anti-CTLA-4×anti-ICOS

Anti-CTLA-4×anti-ICOS bottle-openers were generated with an anti-ICOSFab derived from XENP16435 and anti-CTLA-4 scFvs derived from ABDsdescribed in Example 2B. Sequence for an exemplary anti-ICOS×anti-CTLA-4bottle-opener is depicted in FIG. 63. Bispecific mAbs were produced asgenerally described in Example 5I(c).

2. 6B: Anti-LAG-3×Anti-ICOS

Anti-LAG-3×anti-ICOS bispecific antibodies were generated withanti-LAG-3 Fabs derived from ABDs described in Example 2C and ananti-ICOS Fab derived from XENP16435. Sequences for exemplaryanti-LAG-3×anti-ICOS bispecific antibodies are depicted in FIG. 64.Bispecific mAbs were produced as generally described in Example 5I(c).

3. 6C: Anti-TIM-3×Anti-ICOS

Anti-TIM-3×anti-ICOS bispecific antibodies were generated withanti-TIM-3 Fabs derived from ABDs described in Example 2D and ananti-ICOS Fab derived from XENP16435. Sequence for an exemplaryanti-TIM-3×anti-ICOS bispecific antibody is depicted in FIG. 65.Bispecific mAbs were produced as generally described in Example 5I(c).

4. 6D: Anti-PD-L1×Anti-ICOS

Anti-PD-L1×anti-ICOS bottle-openers were generated with anti-PD-L1 Fabsderived from ABDs described in Example 2E and anti-ICOS scFvs asdescribed in Example 4B. Sequences for exemplary anti-PD-L1×anti-ICOSbispecific antibodies are depicted in FIG. 66. The bottle-openers wereproduced as generally described in Example 5A.

5. 6E: Additional Costim×Checkpoint Blockade Bispecific AntibodiesFunction to Enhance Cytokine Secretion

Human PBMCs were stimulated with 200 ng/mL SEB for 2 days. Cells werewashed twice then restimulated with 100 ng/mL SEB and treated with theindicated concentrations of the indicated test articles (as describedabove). 24 hours after treatment, supernatants were assayed for IL-2.The data are depicted in FIG. 67 and show that each of the additionalcostim×checkpoint blockade bispecific antibodies enhance IL-2 secretionin comparison to bivalent anti-RSV (XENP15074) control. Notably, themajority of these alternative checkpoint blockade antibodies (but notall, e.g. TIM-3 blockade) enhance IL-2 secretion in comparison to abivalent anti-PD-1 antibody (XENP16432).

G. Example 7: Monovalent Ligation of ICOS is Superior to BivalentCrosslinking

In Example 5A(a), it was surprisingly found that anti-ICOS×anti-PD-1antibody worked to enhance cytokine secretion despite monovalent bindingof ICOS which is contrary to the crosslinking of costimulatory receptorssuch as ICOS which was thought to be necessary for stimulation ofcytokine production. In Example 5H(a), it was surprisingly found thatanti-ICOS×anti-PD-1 antibodies enhanced AKT phosphorylation (a signatureof ICOS agonism) over bivalent anti-ICOS mAbs. In this section, wefurther examine this trend in which monovalent binding of ICOS appearsto be superior to bivalent binding of ICOS.

1. 7A: Production of One-Arm Anti-ICOS Fab-Fc Antibodies

Amino acid sequences for illustrative one-arm anti-ICOS Fab-Fcantibodies are listed in FIG. 68. DNA encoding the three chains neededfor antibody expression were generated by gene synthesis and weresubcloned using standard molecular biology techniques into theexpression vector pTT5. DNA was transfected into HEK293E cells forexpression and resulting proteins were purified using standardtechniques.

The resultant one-arm anti-ICOS Fab-Fc antibodies were characterized foraffinity to human ICOS using Octet. Anti-mouse Fc (AMC) biosensors wereused to capture mouse IgG2a Fc fusion of ICOS and dipped into multipleconcentrations of the test articles. The resulting equilibriumdissociation constants (K_(D)), association rates (ka), and dissociationrates (kd) are presented in FIG. 69. Binding affinities and kinetic rateconstants were obtained by analyzing the processed data using a 1:1binding model using ForteBio Octet Data Analysis software (ForteBio).

2. 7B: Monovalent One-Arm Anti-ICOS Fab-Fc Antibodies Promote GreaterAKT Activation in PBMCs than Bivalent Anti-ICOS Antibody

PBMCs were stimulated with 100 ng/mL SEB for 2 days. Followingstimulation, CD3+ T cells were isolated by negative selection usingEasySep™ Human T Cell Enrichment Kit (STEMCELL Technologies, Vancouver,Canada) and then treated with indicated test articles in combinationwith plate bound anti-CD3 antibody (OKT3; 500 ng/mL). Cells were lysed30 minutes after treatment and assayed for total AKT and phosphorylatedAKT (Ser473) by a multiplexed phosphoprotein assay on MULTI-SPOT384-Well Spot plates (Meso Scale Discovery, Rockville, Md.). The dataare depicted in FIG. 70 as percentage of AKT phosphorylated followingtreatment.

Consistent with Example X, the anti-ICOS×anti-PD-1 bispecific antibodiespromoted significantly greater AKT activation than bivalent anti-ICOSantibody. Surprisingly, monovalent one-arm anti-ICOS Fab-Fc antibodiesenhanced also promoted significantly greater AKT activation thanbivalent anti-ICOS antibody to a level comparable to the bispecificantibodies.

3. 7C: Monovalent Agonism of ICOS Works with Multiple Anti-ICOS ABDs

CD3+ T cells were isolated by negative selection using EasySep™ Human TCell Enrichment Kit (STEMCELL Technologies, Vancouver, Canada) and thentreated with indicated test articles in combination with plate boundanti-CD3 antibody (OKT3; 500 ng/mL). Cells were lysed 30 minutes aftertreatment and assayed for total AKT and phosphorylated AKT (Ser473) by amultiplexed phosphoprotein assay on MULTI-SPOT 384-Well Spot plates(Meso Scale Discovery, Rockville, Md.). The data is depicted in FIG. 74and show that monovalent anti-ICOS Fab-Fc antibodies comprising variousanti-ICOS ABDs were able to induce AKT phosphorylation.

H. Example 8: Investigating In Vitro Binding by XmAb23104

1. XmAb23104 Binding to SEB-Stimulated T Cells

Staphylococcal Enterotoxin B (SEB) is a superantigen that causes T cellactivation and proliferation in a manner similar to that achieved byactivation via the T cell receptor (TCR), including expression ofcheckpoint receptors such as PD-1. Accordingly, human PBMCs from 3separate donors were stimulated with 100 ng/mL SEB for 3 days. Cellswere then treated with the following test articles at the indicatedconcentrations for 30 minutes: XmAb23104; XENP20111, a one-armed scFv-Fcbased on the anti-PD-1 arm from XmAb23104; and XENP24901, a one-armedFab-Fc based on the anti-ICOS arm from XmAb23104. Following incubation,cells were washed and stained with anti-human-Fc-A647 and anti-CD3-FITC.Cells were washed two more times and assayed by FACS. MFI on CD3+ Tcells indicating binding of the test articles are depicted in FIGS.90A-D, respectively for each donor.

The data show that, in PBMCs from each of the donors, XmAb23104 bindsmore avidly to CD3⁺ T cells compared to one-armed controls XENP20111 andXENP24901, demonstrating that binding to human T cells is significantlybetter by bispecific antibody XmAb23104, where each arm monovalentlybinds a different antigen, than by monovalent, monospecific antibodies.

2. Occupancy of PD-1 and ICOS on SEB-Stimulated T Cells by XmAb23104

Receptor occupancy by XmAb23104 was measured in an SEB-stimulated PBMCassay. Human PBMCs were stimulated with 500 ng/mL SEB for 3 days. Cellswere then treated with the following test articles at indicatedconcentrations for 30 minutes: XmAb23104; XENP20111; XENP24901;XENP19686, a bivalent mAb based on the anti-PD-1 arm from XmAb23104;XENP16435, a bivalent mAb based on the parental clone from which theanti-ICOS arm of XmAb23104 was generated; and XENP15074, a bivalentanti-RSV mAb used as a control. Following incubations, cells werepelleted and stained with A488-conjugated XENP20111, A647-conjugatedXENP24901, and BV510-conjugated anti-CD3 mAb. Cells were then washed twotimes and assayed by FACS. FIGS. 91A-D show receptor occupancy followingtreatment with the various test articles as depicted by percentage ofvarious populations of CD3⁺ T cells with unoccupied PD-1 and/or ICOSreceptors as indicated by staining. For example, occupancy of PD-1receptors decreases the percentage of ICOS⁺PD-1⁺ and ICOS⁻PD-1⁺populations and increases the percentage of ICOS⁺PD-1⁻ and ICOS⁻PD-1⁻populations. FIGS. 92A-B respectively show the amount of unoccupied ofPD-1 and ICOS receptors on ICOS⁺PD-1⁺CD3⁺ T cells following treatmentwith test articles as indicated by XENP20111 and XENP24901 binding.

I. Example 9: XmAb23104 Exhibits a Multi-Gene Expression SignatureConsistent with ICOS Costimulation

FIG. 93 depicts the fold change in expression of additional genes (asdetermined by Nanostring nCounter® in the experiment described inExample 5H(b)) between XmAb23104, anti-PD-1 mAb (XENP19686), anti-ICOSmAb (XENP16435), anti-PD-1 mAb (XENP19686) in combination with anti-ICOSmab (XENP16435), and negative control anti-RSV mAb (XENP15074).

J. Example 10: XmAb23104 Enhances Anti-Tumor Responses in Mice

NOD SCID gamma (NSG) mice were engrafted intradermally with either 3×10⁶pp65-expressing MCF-7 cells or 2×10⁶ pp65-expressing MDA-MB-231 cells inthe rear flank on Day −14. On Day 0, mice were engraftedintraperitoneally with 5×10⁶ human PBMCs from a CMV⁺ donor that screenedpositive for T cell pp65 reactivity. Mice were treated weekly withXmAb23104 (control mice were dosed with PBS) for 4 weeks (or 4 totaldoses). Tumor volumes were monitored by caliper measurements and dataare shown (days post 1^(st) dose) in FIGS. 94 and 95.

K. Example 11: Further In Vitro Characterization of XmAb23104

1. 11A: XmAb23104 Blocks PD-1 Binding by PD-L1 and PD-L2

HEK293T cells stably expressing ICOS (Crown Bioscience, Santa Clara,Calif.) were stably transfected with a pCMV6-AC-GFP vector encoding PD-1(OriGene, Rockville, Md.). Cells were treated with the following testarticles for 30 minutes at 4° C.: XmAb23104, XENP16432 (a bivalentanti-PD-1 mAb based on nivolumab), XENP20111 (a monovalent anti-PD-1scFv-Fc fusion based on the anti-PD-1 arm from XmAb23104), and XENP24901(a monovalent anti-ICOS Fab-Fc fusion based on the anti-ICOS arm fromXmAb23104 with M428L/N434S Xtend Fc; sequence depicted in FIG. 1). Cellswere washed and incubated with A647-conjugated PD-L1-Fc (1 μg/mL;sequence depicted in FIG. 2A) or A647-conjugated PD-L2-Fc (0.1 μg/mL;sequence depicted in FIG. 2B) for 30 minutes at 4° C. Cells were washedtwice more and analyzed by flow cytometry. PD-L1 and PD-L2 binding (asindicated by A647 MFI on GFP+ cells) following treatment with theindicated test articles are depicted respectively in FIGS. 3A-B. Thedata show that XmAb23104, XENP16432, and XENP20111 blocked PD-L1 andPD-L2 binding to PD-1⁺ICOS⁺ cells in a dose dependent manner, whileXENP24901 did not block PD-L1 and PD-L2 binding.

2. 11B: XmAb23104 Blocks ICOS Binding by ICOSL

HEK293T cells stably expressing ICOS (Crown Bioscience, Santa Clara,Calif.) were stably transfected with a pCMV6-AC-GFP vector encoding PD-1(OriGene, Rockville, Md.). Cells were treated with the following testarticles for 30 minutes at 4° C.: XmAb23104, XENP20111, and XENP24901.Cells were washed and incubated with A647-conjugated ICOSL-Fc (0.5μg/mL; sequence depicted in FIG. 4) for 30 minutes at 4° C. Cells werewashed twice more and analyzed by flow cytometry. ICOSL binding (asindicated by A647 MFI on GFP+ cells) following treatment with theindicated test articles is depicted in FIG. 5. The data show thatXmAb23104 and XENP24901 blocked ICOSL binding to PD-1⁺ICOS⁺ cells in adose dependent manner, while XENP20111 did not block ICOSL binding.

3. 11C: XmAb23104 Binds Cynomolgus T Cells

Towards preclinical studies, it is helpful to have antibodies that arecross-reactive for human and cynomolgus monkeys. Accordingly, weconfirmed whether XmAb23104 (and its component arms) could bindcynomolgus T cells. PBMCs collected from 4 cynomolgus monkeys werestimulated with 100 ng/mL SEB for 3 days. Cells were then treated withthe following test articles at the indicated concentrations: XmAb23104,XENP20111, and XENP24901. Following incubation, cells were washed andstained with anti-human-Fc-A647 and anti-CD3-FITC. Cells were washed twomore times and assayed by FACS. MFI on CD3⁺ T cells indicating bindingof the test articles are depicted in FIGS. 6A-D respectively for eachanimal. The data show that XmAb23104, as well as its component Fab andscFv arms as Fc-fusions, bound to CD3⁺ cells from cynomolgus monkeys.

4. 11D: XmAb23104 Enhances IFNγ and IL-2 Secretion in an SEB-StimulatedPBMC Assay

Human PBMCs from 21 donors were stimulated with 100 ng/mL SEB for 2days. Cells were washed two times and re-stimulated with 100 ng/mL SEBand 20 μg/mL of the following test articles for 24 hours: XENP16432,XENP16435 (a bivalent mAb based on the parental clone from which theanti-ICOS arm of XmAb23104 was generated), XENP20111, XENP24901,XENP16432 in combination with XENP16435, XENP20111 in combination withXENP24901, and XmAb23104 (as well as XENP15074 as control). Followingincubation with the test articles, cell supernatants were collected andassayed for IFNγ and IL-2. Data depicting IFNγ secretion are depicted inFIG. 7A, and data depicting IL-2 secretion are depicted in FIGS. 7B and8. The data show that XmAb23104 is superior at induction of IFNγ andIL-2 secretion in comparison to a bivalent anti-PD-1 mAb based onnivolumab.

5. 11E: XmAb23104 Enhances IFNγ Secretion in an MLR Assay

The ability of XmAb23104 to enhance IFNγ secretion was investigated in amixed-lymphocyte reaction (also known as a mixed-lymphocyte reaction orMLR). Mixing lymphocytes from unique donors induces allogeneic T cellactivation and proliferation, and subsequently upregulation ofcheckpoint receptors such as PD-1. Human PBMCs from 11 unique donorswere mixed to generate 22 unique MLR mixes (400,000 cells/donor in atotal volume of 100 μL) and treated with 20 μg/mL of the following testarticles for 5 days: XENP16432, XENP19686 (a bivalent anti-PD-1 mAbbased on the anti-PD-1 arm from XmAb23104), XENP16435, XENP21622 (amonovalent anti-PD-1 scFv-Fc fusion based on the anti-PD-1 arm fromXmAb23104 with M428L/N424S Xtend Fc; sequence depicted in FIG. 9),XENP20266 (a monovalent anti-ICOS Fab-Fc fusion based on the parentalclone from which the anti-ICOS arm of XmAb23104 was generated),XENP16432 in combination with XENP16435, XENP21622 in combination withXENP20266, or XmAb23104 (and XENP15074 as control). Followingincubation, cell supernatants were collected and assayed for IFNγ. Datadepicting induction of IFNγ by indicated test articles over controlanti-RSV mAb (XENP15074) are shown in FIG. 10. Consistent with Example1D, the data show that XmAb23104 is superior at induction of IFNγsecretion in comparison to a bivalent anti-PD-1 mAb based on nivolumab.

6. 11F: XmAb23104 Enhanced Cytokine Secretion is Dose-Dependent Manner

Human PBMCs from 2 donors were stimulated with 100 ng/mL SEB for 2 days.Cells were then washed two times and re-stimulated with 100 ng/mL SEB incombination with XmAb23104 or a combination of XENP20111 and XENP24901(at equimolar PD-1 and ICOS binding concentrations relative toXmAb23104). 24 hours after treatment, cell supernatant was collected andassayed for IFNγ and IL-2, as depicted respectively in FIGS. 11-12.

L. Example 12: XmAb23104 in Combination with Anti-CTLA-4 mAb

Next we investigated the effect of combining XmAb23104 with anadditional checkpoint inhibitor. Human PBMCs from 22 donors werestimulated with 100 ng/mL SEB for 2 days. Cells were washed two times,and re-stimulated with 100 ng/mL SEB in combination with 20 μg/mLXmAb23104 alone, XENP16433 (a bivalent anti-CTLA-4 mAb based onipilimumab; sequence depicted in FIG. 13), or XmAb23104 in combinationwith XENP16433 (and XENP15074 as a control). Cell supernatant was thencollected and assayed for IFNγ and IL-2, as depicted respectively inFIGS. 14A-B. The data show that the combination of XmAb23104 withXENP16433 induced greater cytokine secretion than each antibody alone.

M. Example 13: XmAb23104 Enhances Greater T Cell Activation In Vitrothan αPD-1 Alone as Indicated by IL-2 Secretion

21 unique MLR reactions were treated with 20 μg/mL of XmAb23104,XENP16432 (a bivalent mAb based on nivolumab with ablated effectorfunction), or XENP15074 (a bivalent anti-RSV mAb as isotype control) for6 days. Cell supernatants were collected and assayed on MULTI-SPOT384-Well Spot plates (Meso Scale Discovery, Rockville, Md.) for IL-2concentration. Data showing fold change in IL-2 secretion are depictedin FIG. 310, and show that XmAb23104 enhances greater T cell activationthan anti-PD-1 mAb alone.

N. Example 14: XmAb23104 Induces Greater Human Leukocyte Expansion andGVHD in huPBMC-Engrafted NSG Mice than αPD-1 Alone

NSG mice (10 per group) were engrafted via IV-OSP with 10×10⁶ humanPBMCs on Day −1. On Days 0, 7, 14, and 21, mice were dosed withXmAb23104 or XENP16432. Mice were weighted on Days −2, 3, 6, 10, 13, 18,21, 24, and 27, and blood was drawn on Days 8, 14, 21 and 28 to countleukocyte populations. and CD45⁺cell counts on Day 8 are depicted inFIG. 111, and changes in body weight over time (as a percentage ofinitial body weight on Day −2) are depicted in FIG. 112. The data showthat XmAb23104 induced greater leukocyte expansion and enhanced GVHD inhuPBMC-engrafted NSG mice in comparison to αPD-1 alone.

O. Example 15: XmAb23104 Enhances Allogeneic Anti-Tumor Response in NSGMice

NSG mice (10 per group) were intradermally inoculated with 3×106pp65-transduced MCF-7 cells on Day −14. Mice were then intraperitoneallyinjected with 5×106 pp65-reactive human PBMCs (or PBS for control) andtreated with the indicated test articles on Day 0, and further treatedwith the indicated test articles on Days 7, 14, and 21. CD45+ cellcounts on Day 21 post-huPBMC engraftment are depicted in FIG. 113. Tumorvolume on Days 21 and 38 post-huPBMC engraftment are depicted in FIGS.114 and 115. Tumor volume as a time course over the duration of thestudy are depicted in FIG. 116. The data show that XmAb23104 enhancesallogeneic anti-tumor response in NSG mice. Notably, treatment withanti-PD-1×anti-ICOS bispecific XmAb23104 is more efficacious than PD-1blockade alone (using a bivalent anti-PD-1 mAb).

P. Example 16: Costim×Checkpoint Blockade Bispecific Antibodies areHighly Active In Vivo

Here we show an earlier GVHD study investigating prototypeanti-PD-1×anti-ICOS XENP20896 and prototype dual checkpoint inhibitorbispecific antibody XENP20053 (blocking PD-1 and CTLA-4 checkpointreceptors; sequences for which are depicted in FIG. 117) in a GVHDstudy.

NSG mice (10 per group) were injected with PBMC on Day 0, and thentreated with anti-PD-1 mAb, anti-PD-1 mAb in combination withanti-CTLA-4 mAb, anti-PD-1×anti-CTLA-4 bispecific XENP20053, oranti-PD-1×anti-ICOS bispecific XENP20896 on Days 1, 8, 15, 22, and 29.CD45+ cell counts on Day 14 post-huPBMC engraftment are depicted in FIG.118. The data show that anti-PD-1×anti-ICOS bispecific XENP20896 ishighly active in vivo, and enhances engraftment in comparison toanti-PD-1 blockade alone with an anti-PD-1 antibody, dual checkpointblockade with anti-PD-1 and anti-CTLA-4 antibodies, as well as dualcheckpoint blockade with an illustrative anti-PD-1×anti-CTLA-4bispecific antibody.

Q. Example 17: XmAb23104 does not Induce Cytokine Release in Naive TCells and is not Superagonistic

PBMCs were thawed overnight and treated with 20 μg/mL of indicatedsoluble or plate bound test articles for 24 hours. Anti-CD3 antibody wasclone OKT3. Cell supernatants were then collected and assayed withV-PLEX Proinflammatory Panel 1 Human Kit (Meso Scale, Rockville, Md.).Each point represents a unique human donor tested in technical singlet.Paired t tests were used to determine statistical significance (n.s.signifies a p-value >0.05). The data depicted in FIG. 119 show thatXmAb23104 does not induce cytokine release (A: IFNγ; B: IL-1B; C: IL-2;D: IL-4; E: IL-8; F: IL-6; G: IL-10; H: IL-12p70; I: IL-13; J: TNFα) innaive T cells.

Superagonstic properties of XmAb23104 was also assessed by air-dryingper the Stebbings protocol (Stebbings R. et al. 2007). Air-drying oftest articles was achieved by drying in a SpeedVac™ for 2 hours at roomtemperature. Human PBMCs were treated for 24 hours with 10 μg ofair-dried XmAb23104, and activity was compared to 10 μg of air-driedXENP15074 (anti-RSV negative isotype control), the superagonist TGN1412(XENP29154; sequences for which are depicted in FIG. 120), or anti-CD3(OKT3). TGN1412 did not possess any activity when bound to the assayplate using an aqueous adsorption method; however, air-dried TGN1412 (aswell as anti-CD3 mAb) promoted IFNγ, IL-10, IL-2, IL-4, IL-6, IL-8,IL-10, IL-12p70, IL-13, and TNF cytokine secretion from unstimulatedhuman PBMC. In comparison, the cytokine levels in PBMCs treated withair-dried XmAb23104 remained similar to the negative control ofair-dried XENP15074 (data shown in FIG. 121).

R. Example 18: Further Characterization of Binding by XmAb23104

1. A: XmAb23104 Binds Human and Cynomolgus PD-1 and ICOS

Binding of XmAb23104 to human and cynomolgus PD-1 and ICOS wascharacterized using Octet, a BioLayer Interferometry (BLI)-based method.Binding affinities were obtained by analyzing the processed dataglobally using a 1:1 binding model. Octet sensorgrams are shown in FIG.128 and FIG. 129. The resulting equilibrium dissociation constants(K_(D)), association rate constants (k_(a)), and dissociation constants(k_(d)) are presented in FIG. 130. Affinities for both human andcynomolgus PD-1 were measured at approximately 2.8 and 5.8 nMrespectively. Binding affinities for human and cyno ICOS were 2.9 and2.6 nM respectively.

2. B: XmAb23104 Competes for Binding with Ligands of PD-1 and ICOS

Binding of PD-L1 and PD-L2 to PD-1 with and without XmAb23104 wascharacterized using Octet, a BioLayer Interferometry (BLI)-based method.Octet sensorgrams are shown in FIG. 132. In both cases, 100 nM PD-1 showa binding signal with their ligands (PDL1/PDL2). In the presence ofexcess XmAb23104, pre-incubated with PD-1 at room temperature for 1 hourprior to the experiment, there is no binding signal observed to anyligands due to the competition of XmAb23104 with PD-1 for its ligandsPD-L1 and PD-L2.

In another experiment to investigate the competition of XmAb23104 withICOS ligand (ICOSL) and well as with PD-1 ligands (PD-L1 and PD-L2), HEK293T cells expressing PD-1 and ICOS were incubated with variousconcentrations of XmAb23104 for 30 minutes at 4° C. Followingincubation, 1 μg/mL PD-L1-mFc-Alexa647, 0.1 μg/mL PD-L1-mFc-Alexa647, or0.5 μg/mL ICOSL-mFc-Alex647 was added and incubated for 30 minutes at 4°C. Binding of soluble ligands was visualized with flow cytometry. Dataare depicted in FIG. 119, and show that XmAb23104 blocked binding ofsoluble PD-L1 (EC₁₀ 1 ng/mL and EC₉₀ 7100 ng/mL) and PDL2 (EC₁₀ 3 ng/mLand EC₉₀ 5300 ng/mL) antigen binding to cell surface-expressed PD1receptor antigen in a dose-dependent fashion, as well as binding ofsoluble ICOSL (EC₁₀ 20 ng/mL, EC₉₀ 600 ng/mL) to cell surface-expressedICOS receptor in a dose-dependent fashion.

3. C: XmAb23104 does not Bind FcγR

Binding of XmAb23104 to human, cynomolgus, and mouse FcγRs wascharacterized using Octet, a BioLayer Interferometry (BLI)-based method.A comparator antibody was also tested using similar methods: anti-CD19antibody with a native IgG1 constant region. Octet sensorgrams are shownin the FIG. 127 to FIG. 129. While the expected binding patterns for anative human IgG1 antibody were observed for the comparator antibody, nobinding for any of the FcγRs was detected for XmAb23104.

4. D: XmAb23104 Binds Human, Cynomolgus, and Mouse FcRn at pH 6.0

Binding of XmAb23104 to human, cynomolgus, and mouse FcRn at pH 6.0 wascharacterized using Octet, a BioLayer Interferometry (BLI)-based method.A comparator antibody was also tested using similar methods: XENP20896,an anti-PD1×anti-ICOS bispecific antibody containing the same variableregions and engineered constant regions as XmAb23104 but lacking theamino acid substitutions XmAb23104 contains for enhancing FcRn binding.Binding affinities were obtained by analyzing the processed dataglobally using a 1:1 Langmuir model. Octet sensorgrams are shown in FIG.131. The resulting equilibrium dissociation constants (K_(D)) arepresented Figure. Affinities measured for XmAb23104 are tighter thanthose measured for the comparator, indicating that the Fc substitutionscontained in XmAb23104 improve the affinity for FcRn at pH 6.0, thephysiologically relevant pH for endosome trafficking.

5. E: XmAb23104 Simultaneously Binds PD-1 and ICOS

Binding of XmAb23104 to both ICOS and PD1 antigens was tested using anin-tandem dip approach using BLI technology on the Octet HTX instrument.First, biosensors were loaded with ICOS, then dipped into eitherXmAb23104 or buffer as a control, and finally, into PD-1. FIG. 132 showsthe binding sensorgrams which indicate that XmAb23104 binds to bothantigens simultaneously. The XmAb23104 sensorgram continues to increasein signal during the final PD-1 antigen dip while the control sensorgramwith no XmAb23104 loaded remains flat.

S. Example 19: Further In Vitro Characterization of XmAb23104

1. A: XmAb23104 Dose-Dependently Promoted IL-2 and IFNγ Secretion fromSEB-Stimulated Human PBMC

IL-2 and IFNγ production was assessed in SEB-stimulated PBMC treatedwith soluble XmAb23104. Human PBMC (100,000) were cultured in RPMI1640with 10% FBS and stimulated with 100 ng/mL SEB (48 hours or 96 hours;37° C.). Logistical considerations necessitated two sets ofpre-stimulation conditions; of note, 96 hours of pre-stimulationabrogates detection of IL-2. Following stimulation, cells were washedtwo times in warm culture medium and then cultured in medium containingthe indicated amount of XmAb23104 plus 100 ng/mL SEB (18 hours; 37° C.).To evaluate cytokine release, culture supernatant was collected 18 hoursafter treatment, then assayed for IL-2 and IFNγ secretion by ELISA (MSD®format) per the manufacturer's instructions. Samples were assayed intechnical singlet.

Soluble XmAb23104 promoted a dose-dependent increase in IL-2 and IFNγ inall 19 donors tested with variable maximum amounts of cytokineexpression induced between donors varying approximately between 1 ng/mLto 10 ng/mL for IL-2 (FIG. 133) and from 5 ng/mL to 25 ng/mL for IFNγ(FIG. 134 The potency was derived from curve fits, with least-squaresmethods all providing acceptable curve fits from which the EC valueswere derived. The average potency of XmAb23104 to induce IL-2 releasefrom SEB-stimulated PBMC is: EC₁₀ 44 ng/mL, EC₂₀ 79 ng/mL, EC₃₀ 125ng/mL, EC₅₀ 273 ng/mL, EC₈₀ 1201 ng/mL, and EC₉₀ 3215 ng/mL. The averagepotency of XmAb23104 in stimulating IFNγ release from humanSEB-stimulated PBMC is: EC₁₀ 21 ng/mL, EC₂₀ 34 ng/mL, EC₃₀ 48 ng/mL,EC₅₀ 81 ng/mL, EC₈₀ 206 ng/mL, and EC₉₀ 384 ng/mL.

2. B: XmAb23104 Dose-Dependently Promotes IFNγ Secretion fromSEB-Stimulated Cynomolgus PBMC

Because IFNγ release was the most sensitive cytokine induced byXmAb23104 in human PBMC, we also quantified IFNγ secretion fromSEB-stimulated PBMC from 10 cynomolgus monkeys. Cynomolgus PBMC(400,000) were cultured in RPMI1640 with 10% FBS and stimulated with 100ng/mL SEB. Following stimulation, cells were washed two times in warmculture medium and then cultured in medium containing the indicatedamount of XmAb23104 plus 100 ng/mL SEB (18 hours; 37° C.). To evaluatecytokine release, culture supernatant was collected 18 hours aftertreatment and assayed for IFNγ secretion by ELISA (MSD® format) per themanufacturer's instructions. Samples were assayed in technical singlet,data for which are depicted in FIG. 135.

The potency of XmAb23104 was derived from the five out of 10 monkey PBMCthat generated data that could be fit to a sigmoidal dose responsecurve. XmAb23104 stimulated IFNγ release from monkey PBMC with thefollowing average effective potencies: EC10 32 ng/mL, EC20 45 ng/mL,EC30 56 ng/mL, EC50 81 ng/mL, EC80 155 ng/mL, and EC90 233 ng/mL. Forcomparison, the EC50 for IFNγ release from SEB stimulated human PBMC was81 ng/mL, which has similar potency to the monkey SEB stimulated PBMC.

T. Example 20: XmAb23104 Anti-Tumor Response is Dose-Dependent

In the study described in Example 15 investigating the enhancement ofallogeneic anti-tumor response in NSG mice by XmAb23104, treatment withadditional concentrations of XmAb23104 was investigated. The datadepicted in FIGS. 114-116 show treatment with 5.0 mg/kg of XmAb23104. InFIG. 136 and FIG. 137, we depict data additionally showing treatmentwith 0.3 mg/kg and 1.0 mg/kg of Xmab23104.

U. Example 21: XmAb23104 Enhances Induction of Leukocyte Expansion inhuPBMC-Engrafted NSG Mice in Comparison to Comparator Anti-ICOS mAb

Since JTX-2011 is being administered to patients in combination withnivolumab, the leukocyte expanding activity of XmAb23104 was compared tothe activity of JTX-2011 alone (in-house produced as XENP23058;sequences depicted in FIG. 138) and in combination with XENP16432(anti-PD-1 mAb based on nivolumab with E233P/L234V/L235A/G236del/S267Kablation variants) in a mouse GVHD study. We also investigated theleukocyte expanding activity of XENP23057 (anti-ICOS mAb based onJTX-2011 with E233P/L234V/L235A/G236del/S267K ablation variants;sequences depicted in FIG. 139) alone and in combination with XENP16432.NSG mice were engrafted with 5×10⁶ huPBMCs on Day −1, followed by dosingwith the indicated test articles at indicated concentrations on Days 0,7, 14, and 21. Mice were bled and whole blood analyzed to investigatethe expansion of various human lymphocyte populations, data for whichare depicted in FIG. 140 for Day 21.

Notably, the data show that JTX-2011 (XENP23058), alone and incombination with XENP16432, decreased human leukocytes in comparison toinduction of leukocyte expansion by XENP16432 and XmAb23104. We reasonedthat this is due to the WT IgG1 backbone of XENP23058 which induceddepletion of ICOS positive cells by ADCC or CDC through binding ofactivating FcγR. Consistent with this reasoning, XENP23057 which hasablated FcγR binding, alone and in combination with XENP16432, inducedexpansion of human leukocytes.

1. A bispecific antibody that monovalently binds a human costimulatoryreceptor and monovalently binds a human checkpoint receptor for use inactivating T cells for the treatment of cancer.
 2. A bispecific antibodyaccording to claim 1 wherein said costimulatory receptor is selectedfrom the group consisting of ICOS, GITR, OX40 and 4-1BB.
 3. A bispecificantibody according to claim 1 or 2 wherein said checkpoint receptor isselected from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3 andTIM-3.
 4. A bispecific antibody according to claim 1, 2 or 3 whereinsaid costimulatory receptor is human ICOS.
 5. A bispecific antibodyaccording to claim 4 wherein said antibody binds ICOS and PD-1.
 6. Abispecific antibody according to claim 4 wherein said antibody bindsICOS and PD-L1.
 7. A bispecific antibody according to claim 4 whereinsaid antibody binds ICOS and CTLA-4.
 8. A bispecific antibody accordingto claim 4 wherein said antibody binds ICOS and LAG-3.
 9. A bispecificantibody according to claim 4 wherein said antibody binds ICOS andTIM-3.
 10. A bispecific antibody according to any previous claim whereinsaid antibody has the format of FIG. 2A.
 11. A bispecific antibodyaccording to any previous claim wherein said antibody has the format ofFIG. 2E.
 12. A bispecific antibody according to any previous claimwherein said antibody has the format of FIG. 2F.
 13. A bispecificantibody according to any previous claim wherein said antibody has theformat of FIG. 2G.
 14. A bispecific antibody according to claim 5wherein said antibody comprises: a) a first sequence having SEQ IDNO:26642; b) a second sequence having SEQ ID NO:26657; and c) a thirdsequence having SEQ ID NO:26647.
 15. A heterodimeric antibodycomprising: a) a first heavy chain comprising a first Fc domain, anoptional domain linker and a first antigen binding domain comprising anscFv that binds a first antigen; b) a second heavy chain comprising aheavy chain comprising a heavy chain constant domain comprising a secondFc domain, a hinge domain, a CH1 domain and a variable heavy domain; andc) a light chain comprising a variable light domain and a light chainconstant domain; wherein said variable heavy domain and said variablelight domain form a second antigen binding domain that binds a secondantigen, wherein one of said first and second antigen binding domainsbinds human ICOS and the other binds human PD-1.
 16. A heterodimericantibody comprising: a) a first heavy chain comprising: i) a firstvariant Fc domain; and ii) a single chain Fv region (scFv) that binds afirst antigen, wherein said scFv region comprises a first variable heavychain, a variable light chain and a charged scFv linker, wherein saidcharged scFv linker covalently attaches said first variable heavy chainand said variable light chain; and b) a second heavy chain comprising aVH—CH1-hinge-CH2-CH3 monomer, wherein VH is a second variable heavychain and CH2-CH3 is a second variant Fc domain; and c) a light chain;wherein said second variant Fc domain comprises amino acid substitutionsN208D/Q295E/N384D/Q418E/N241D, wherein said first and second variant Fcdomains each comprise amino acid substitutionsE233P/L234V/L235A/G236del/S267K; and wherein said first variant Fcdomain comprises amino acid substitutions S364K/E357Q and second variantFc domain comprises amino acid substitutions L368D/K370S, wherein one ofsaid first and second antigen binding domains binds human ICOS and theother binds human PD-1, and wherein numbering is according to the EUindex as in Kabat.
 17. A heterodimeric antibody according to anyprevious claim wherein said first and second variant Fc domains eachcomprise M428L/N434S.
 18. A heterodimeric antibody comprising: a) afirst heavy chain comprising: i) a first variant Fc domain; and ii) asingle chain Fv region (scFv) that binds a first antigen, wherein saidscFv region comprises a first variable heavy chain, a variable lightchain and a charged scFv linker, wherein said charged scFv linkercovalently attaches said first variable heavy chain and said variablelight chain; and b) a second heavy chain comprising aVH—CH1-hinge-CH2-CH3 monomer, wherein VH is a second variable heavychain and CH2-CH3 is a second variant Fc domain; and c) a light chain;wherein said first and second variant Fc domains comprises a set ofheterodimerization variants selected from the group consisting ofL368D/K370S: S364K/E357Q; L368D/K370S: S364K; L368E/K370S: S364K;T411E/K360E/Q362E: D401K; and T366S/L368A/Y407V: T366W, and wherein oneof said first and second antigen binding domains binds human ICOS andthe other binds human PD-1, and wherein numbering is according to the EUindex as in Kabat.
 19. A nucleic acid composition comprising: a) a firstnucleic acid encoding a first heavy chain according to any of claims 1to 18; b) a second nucleic acid encoding a second heavy chain accordingto any of claims 1 to 18, respectively; and c) a third nucleic acidencoding a light chain according to any of claims 1 to 18, respectively.20. An expression vector composition comprising: a) a first expressionvector comprising said first nucleic acid of claim 19; b) a secondexpression vector comprising said second nucleic acid of claim 19; andc) a third expression vector comprising said third nucleic acid of claim19.
 21. A host cell comprising the expression vector composition ofclaim
 20. 22. A method of making a heterodimeric antibody comprisingculturing a host cell of claim 21 under conditions wherein saidheterodimeric antibody is expressed, and recovering said antibody.
 23. Amethod of activating cytotoxic T cells (CTLs) in a patient comprisingadministering a heterodimeric antibody according to any of claims 1 to18 to said patient.
 24. A method of increasing IFNγ levels in a patientcomprising administering a heterodimeric antibody according to any ofclaims 1 to 18 to said patient.
 25. A method of increasing IL-2 levelsin a patient comprising administering a heterodimeric antibody accordingto any of claims 1 to 18 to said patient.